Methods and compositions for the manufacture of C-3&#39; and C-4&#39; anthracycline antibiotics

ABSTRACT

The present invention discloses new and novel substituted anthracyclines with modified alkyl-aromatic ring substitutions on the C-3&#39; of the sugar moiety or modified or unmodified alkyl-aromatic ring substitutions at the C-4&#39; of the sugar moiety. It also discloses novel methods for the preparation of sugar substrates and methods for the preparation of anthracycline antibiotics. These anthracycline analogs show high cytotoxicity in vitro against several tumor cell lines.

The U.S. Government may owns rights in the present invention pursuant toNational Institute of Health grants numbered C3A55270 and CA50320.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the treatment of cancer. Moreparticularly, it concerns novel compounds useful for chemotherapy,methods of synthesis of these compounds and methods of treatmentemploying these compounds. These novel drugs comprise two main classesof compounds; one bearing modified substituents at the C-3′ sugar moietyand the other bearing modifications at the C-4′ sugar moiety. Inaddition, some of these analogs might also be modified at the aglyconand/or sugar moiety. These novel anthracycline analogs display highanti-tumor activity and can be used as potent drugs active againstmulti-drug resistant tumors. These compounds are related to otheranti-tumor anthracyclines such as daunorubicin, idarubicin, epirubicin,and doxorubicin. The cytotoxic potency of these new compounds issignificantly higher when compared to doxorubicin.

2. Description of Related Art

Resistance of tumor cells to the killing effects of chemotherapy is oneof the central problems in the management of cancer. It is now apparentthat at diagnosis many human tumors already contain cancer cells thatare resistant to standard chemotherapeutic agents. Spontaneous mutationtoward drug resistance is estimated to occur in one of every 10⁶ to 10⁷cancer cells. This mutation rate appears to be independent of anyselective pressure from drug therapy, although radiation therapy andchemotherapy may give rise to additional mutations and contribute totumor progression within cancer cell populations (Goldie et al, 1979;Goldie et al, 1984; Nowell, 1986). The cancer cell burden at diagnosisis therefore of paramount importance because even tumors as small as 1cm (10⁹ cells) could contain as many as 100 to 1,000 drug-resistantcells prior to the start of therapy.

Selective killing of only the tumor cells sensitive to the drugs leadsto an overgrowth of tumor cells that are resistant to the chemotherapy.Mechanisms of drug resistance include decreased drug accumulation(particularly in multi-drug resistance), accelerated metabolism of thedrug and other alterations of drug metabolism, and an increase in theability of the cell to repair drug-induced damage (Curt et al., 1984;and Kolate, 1986). The cells that overgrow the tumor population not onlyare resistant to the agents used but also tend to be resistant to otherdrugs, many of which have dissimilar mechanisms of action. Thisphenomenon, called pleiotropic drug resistance or multi-drug resistance(MDR), may account for much of the drug resistance that occurs inpreviously treated cancer patients. The development of drug resistanceis one of the major obstacles in the management of cancer. One of thetraditional ways to attempt to circumvent this problem of drugresistance has been combination chemotherapy.

Combination drug therapy is the basis for most chemotherapy employed totreat breast, lung, and ovarian cancers as well as Hodgkin's disease,non-Hodgkin's lymphomas, acute leukemias, and carcinoma of the testes.Combination chemotherapy uses the differing mechanisms of action andcytotoxic potentials of multiple drugs.

Although combination chemotherapy has been successful in many cases, theneed still exists for new anti-cancer drugs. These new drugs could besuch that they are useful in conjunction with standard combinationchemotherapy, or these new drugs could attack drug resistant tumors byhaving the ability to kill cells of multiple resistance phenotypes.

A drug that exhibits the ability to overcome multiple drug resistancecould be employed as a chemotherapeutic agent either alone or incombination with other drugs. The potential advantages of using such adrug in combination with chemotherapy would be the need to employ fewertoxic compounds in the combination, cost savings, and a synergisticeffect leading to a treatment regime involving fewer treatments.

The commonly used chemotherapeutic agents are classified by their modeof action, origin, or structure, although some drugs do not fit clearlyinto any single group. The categories include alkylating agents,anti-metabolites, antibiotics, alkaloids, and miscellaneous agents(including hormones). Agents in the different categories have differentsites of action.

Antibiotics are biologic products of bacteria or fungi. They do notshare a single mechanism of action. The anthracyclines daunorubicin anddoxorubicin (DOX) are some of the more commonly used chemotherapeuticantibiotics. The anthracyclines achieve their cytotoxic effect byseveral mechanisms, including inhibition of topoisomerase II;intercalation between DNA strands, thereby interfering with DNA and RNAsynthesis; production of free radicals that react with and damageintracellular proteins and nucleic acids; chelation of divalent cations;and reaction with cell membranes. The wide range of potential sites ofaction may account for the broad efficacy as well as the toxicity of theanthracyclines (Young et al., 1985).

The anthracycline antibiotics are produced by the fungus Streptomycespeuceitius var. caesius. Although they differ only slightly in chemicalstructure, daunorubicin has been used primarily in the acute leukemias,whereas doxorubicin displays broader activity against human neoplasms,including a variety of solid tumors. The clinical value of both agentsis limited by an unusual cardiomyopathy, the occurrence of which isrelated to the total dose of the drug; it is often irreversible. In asearch for agents with high anti-tumor activity but reduced cardiactoxicity, anthracycline derivatives and related compounds have beenprepared. Several of these have shown promise in the early stages ofclinical study, and some, like epirubicin and idarubicin, are used asdrugs. Epirubicin outsells doxorubicin in Europe and Japan, but it isnot sold in the U.S.

The anthracycline antibiotics have tetracycline ring structures with anunusual sugar, daunosamine, attached by glycosidic linkage. Cytotoxicagents of this class all have quinone and hydroquinone moieties onadjacent rings that permit them to function as electron-accepting anddonating agents. Although there are marked differences in the clinicaluse of daunorubicin and doxorubicin, their chemical structures differonly by a single hydroxyl group on C14. The chemical structures ofdaunorubicin and doxorubicin are shown in FIG. 1.

Doxorubicin's broad spectrum of activity against most hematologicalmalignancies as well as carcinomas of the lung, breast, and ovary hasmade it a leading agent in the treatment of neoplastic disease(Arcamone, 1981; Lown, 1988; Priebe, 1995). Since the discovery ofdaunorubicin and doxorubicin (FIG. 1), the mechanistic details of theanti-tumor activity of anthracycline antibiotics have been activelyinvestigated (Priebe, 1995a; Priebe, 1995b; Booser, 1994).

Unfortunately, concomitant with its anti-tumor activity, DOX can produceadverse systemic effects, including acute myelosuppression, cumulativecardiotoxicity, and gastrointestinal toxicity (Young et al., 1985). Atthe cellular level, in both cultured mammalian cells and primary tumorcells, DOX can select for multiple mechanisms of drug resistance thatdecrease its chemotherapeutic efficacy. These mechanisms includeP-gp-mediated MDR and MPR-rediated MDR, characterized by theenergy-dependent transport of drugs from the cell (Bradley et al.,1988), and resistance conferred by decreased topoisomerase II activity,resulting in the decreased anthracycline-induced DNA strand scission(Danks et al., 1987; Pommier et al., 1986; Moscow et al., 1988).

Among the potential avenues of circumvention of systemic toxicity andcellular drug resistance of the natural anthracyclines is thedevelopment of semi-synthetic anthracycline analogues which demonstrategreater tumor-specific toxicity and less susceptibility to various formsof resistance.

SUMMARY OF THE INVENTION

The present invention seeks to overcome drawbacks inherent in the priorart by providing compositions of agents that display increasedcytotoxicity when compared with doxorubicin and can prevent and/orovercome multi-drug resistance and exhibit reduced cardiotoxicity. Thisinvention involves novel compounds that have utility as anti-tumorand/or chemotherapeutic drugs, methods of synthesizing these compoundsand methods of using these compounds to treat patients with cancer. Theinvention is based on the discovery that anthracycline derivatives withsubstitutions attached to their C-3′ or C-4′ carbons in the sugar moietyhave a surprisingly strong ability to kill multi-drug resistant tumorcells.

New anthracycline-based agents designed to interact and crosslink withDNA have been synthesized. These analogs contain substitutions at theC-3′ or C-4′ sugar moiety. Synthesized compounds displayed activitysignificantly higher than that of parental daunorubicin or doxorubicin.In brief, in vitro the compounds WP755, WP756, WP757, WP758, WP775,WP778, WP784, WP786, WP790, WP791 modified at the C-3′ and WP744, WP783-and WP750 modified at the C4′ were significantly more effective asmeasured by resistance index (RI) (Table 2). The RI values for the3′-O-substituted analogs vary from 1.2 to 36 and are low when comparedto the RI value of 253 and >200 for DOX, wherein, a higher RI valueindicates that a compound is less effective against MDR. Similarly, RIvalues were very low (1.4−D8.8) for 4′-O-alkylated analogs whereas RIvalues for DOX varied from >42.6 to >200 for MDR1 type of resistance andfrom 10 to 16 for the MRP form of resistance. Lower RI values indicategreater efficacy of the drug against MDR tumors. The inventors alsodesigned and synthesized other analogs. The observed activity and highpotency against multi-drug resistant tumors indicate that these analogsare different from the parental drugs like doxorubicin and daunorubicin.

The inventors synthesized a series of analogs substituted at thearomatic ring of the C-3′-substituent which were then combined withmodifications at the aglycon moiety. The inventors discovered thatsubstitution at the aromatic ring increased the potency and alter themechanism of action of the drugs making them significantly more activethan doxorubicin. The mechanism of action of this class of drugs mightinvolve direct interaction of the aromatic ring with cellular targetslike DNA, topoisomerase II and topoisomerase 1. Substitution of a benzylring at C-3′ modifies drug interaction with P-glycoprotein, andsubsequently the resistance index, thereby makes these compounds moreeffective against MDR tumors in comparison to the parent drug. In vitroevaluation identified the following C-3′ substituted anthracyclineanalogs: WP831, WP791, WP790, WP786, WP785, WP784, WP780, WP778, WP775,WP774, WP765, WP758, WP757, WP756 and WP755 as unusually effectivecytotoxic agents when compared to DOX.

The inventors also synthesized anthracycline analogs with substitutionsat the C-4′ sugar and demonstrated that these analogs overcome both, (a)multi-drug resistance (MDR) caused by overexpression of the MDR1 geneand (b) MDR-associated protein (MRP)-related resistance caused byoverexpression of the MRP gene. Such modifications also contribute tothe drugs ability to circumvent others forms of drug resistance andincreased bioavailability. An increased steric hindrance at C-4′ indoxorubicin might reduce drug interaction with P-glycoprotein and MRPand in combination, the increased lipophilicity caused by introductionof aromatic ring further contributes to increase intracellular drugconcentration in MDR cells. Such modifications alter cellular uptake andretention of the drugs without affecting interaction with cellulartargets, which results in cytotoxic effects. In vitro evaluationidentified the following C-4′ substituted anthracycline analogs: WP799,WP797, WP794, WP787, WP783, WP750, WP744, WP727, WP764 and WP571 asunusually potent cytotoxic agents when compared to DOX.

The anthracycline compounds bearing the C-3′ substitutions have amongothers, O-Benzyl, N-Benzyl or S-Benzyl substitutions where the phenyl,an aromatic ring of the benzyl group is substituted. The compoundsbearing C4′ substitutions at the sugar have among others, O-Benzyl,N-Benzyl or S-Benzyl substitutions where the benzyl group is asubstituted or an unsubstituted benzene group. These compounds aredepicted in FIGS. 2-25. These compounds exhibit cytotoxic activitysubstantially different from the activities of doxorubicin ordaunorubicin. These compounds are active against doxorubicin resistanttumors and/or are usually similar or more cytotoxic than doxorubicinagainst sensitive tumors.

In some specific embodiments, the C-3′-substituted anthracyclinecompounds of the present invention have the general formula:

-   -   wherein, R¹ denotes any suitable group or combination of groups        that form but are not limited to a nucleic acid intercalator or        binding compound; a topoisomerase inhibitor, including but not        limited to, an alkyl chain; a (—COCH₂R¹³) group; or a        (—C(OH)—CH₂R¹³); wherein, R¹³ is a hydrogen (—H) group or a        hydroxyl group (—OH); a methoxy group (—OCH₃); an alkoxy group        having 1-20 carbon atoms; an alkyl group having 1-20 carbon        atoms; an aryl group having 1-20 carbon atoms; a fatty acyl        group having the general structure (—O—CO(CH2)_(n)CH₃) wherein        n=an integer from 1 to about 20; or a fatty acyl group having        the general structure (—O—CO(CH2)_(l)(CH═CH)_(m)(CH2)_(n)CH₃)        wherein l is an integer between 1 to 3, m is an integer between        1 and about 6, and n is an integer between 1 to about 9; or a        chain (R) such as —OCO—(CH₂)_(n)—CH₂NH₂ ; or OCO—(CH₂)_(n)—CO₂H        and its salts; each of R² and R³ is, independently of the other,        a hydrogen (—H), a hydroxyl group (—OH); a methoxy group        (—OCH₃); R⁴ is a hydrogen (—H) group; a methoxy group (—OCH₃); a        hydroxyl group (—OH); or a halide; each of Y¹ and Y² is,        independently of the other, a double bonded oxygen, sulphur, or        nitrogen atom; Z is a —H; —OH; a —CO₂H group; or a —CO₂R group;        R⁷, R⁸, are, independently, —H; —OH; a halide; —OR¹⁹; —SH;        —SR¹⁹; —NH₂; —NHR¹⁹; —N(R¹⁹)₂; —CH₃; and R⁷ can additionally be        a saccharide; wherein R¹⁹ is an alkyl chain; an alkylating        moiety; a cycloalkyl chain; a cyclic ring; or a hydrogen; R⁹ can        be —H; —CH₃; alkyl; aryl; CH₂OH, CH₂F; R¹⁰, R¹¹ and R¹² are,        independently, —H; —OH; a halide; —OR; —SH; —SR; —NH₂; —NHR;        —N(R)₂; —CH₃; one of R⁵ and R⁶ is a —H; one of R⁵ and R⁶ is a        X-alkyl-aromatic-ring (AAR) substituent such as —XAAR, wherein,        A is an alkyl group and wherein, AR is an substituted phenyl        ring; or a substituted five-member ring; or a heteroatomic        five-member ring; or a heteroatomic six-member ring such as a        pyridine ring; of the form;    -   wherein, R¹⁴-R¹⁸ are independently a (—H) group; a hydroxyl        group (—OH); a methoxy group (—OCH₃); a nitro group (—NO₂), an        amine group (—NH₂), a halide; an alkoxy group having 1-20 carbon        atoms; an alkyl group having 1-20 carbon atoms; an aryl group        having 1-20 carbon atoms; an alkyl-amino group; an alkyl-thio        group; a cyano group (CN, SCN); an —CO₂H group; an —CO₂R group;        and the aromatic ring may be disubstituted, trisubstituted,        tetrasubstituted or pentasubstituted; and X is a —O, —N or —S,        or —SO, or —SO₂ group; and A is (CH₂)_(n) where n=0, 1, 2, 3, 4,        5, 6, 7, 8, 9, or 10, wherein, if R⁵ is a XAAR substituent R⁶ is        not and if R⁶ is a XAAR substituent R⁵ is not.

In other specific embodiments, the C-4′ substituted anthracyclinecompounds of the present invention have the general formula:

-   -   wherein, R¹ denotes any suitable group or combination of groups        that form but are not limited to a nucleic acid intercalator or        binding compound; a topoisomerase inhibitor, including but not        limited to, an alkyl chain; a (—COCH₂R¹³) group; or a        (C(OH)—CH₂R¹³ ) wherein, R¹³ is a hydrogen (—H) group or a        hydroxyl group (—OH); a methoxy group (—OCH₃); an alkoxy group        having 1-20 carbon atoms; an alkyl group having 1-20 carbon        atoms; an aryl group having 1-20 carbon atoms; a fatty acyl        group having the general structure(—O—CO(CH2)_(n)CH₃) wherein        n=an integer from 1 to about 20; or a fatty acyl group having        the general structure (—O—CO(CH2)_(l)(CH═CH)_(m)(CH2)_(n)CH₃),        wherein 1 is an integer between 1 to 3, m is an integer between        1 and about 6, and n is an integer between 1 to about 9; or a        chain(R) such as —OCO—(CH₂)_(n)—CH₂NH₂ ; or OCO—(CH₂)_(n)—CO₂H        and its salts; each of R² and R³ is, independently of the other,        a hydrogen (—H), a hydroxyl group (—OH); a methoxy group        (—OCH₃); R⁴ is a hydrogen (—H) group; a methoxy group (—OCH₃); a        hydroxyl group (—OH); or a halide; each of Y¹ and Y² is,        independently of the other, a double bonded oxygen, sulphur, or        nitrogen atom; Z is a —H; —OH; a —CO₂H group; or a —CO₂R group;        R⁵, R⁶, are, independently, —H; —OH; a halide; —OR¹⁹; —SH;        —SR¹⁹; —NH₂; —NHR¹⁹; —N(R¹⁹)₂; —CH₃; wherein R¹⁹ is an alkyl        chain; an alkylating moiety; a cycloalkyl chain; a cyclic ring;        or a hydrogen; and R⁶ can additionally be a an alkylating        moiety; R⁹ can be —H; —CH₃; alkyl; aryl; CH₂OH, CH₂F; R¹⁰, R¹¹        and R¹² are, independently, —H; —OH; a halide; —OR; —SH; —SR;        —NH₂; —NHR; —N(R)₂; —CH₃; one of R⁷ and R⁸ is a —H; one of R⁷        and R⁸ is a X-alkyl aromatic-ring (AAR) substituent such as        —XAAR, wherein, A is an alkyl group and wherein, AR is an        unsubstituted phenyl ring; or a substituted phenly ring; or a        substituted five-member ring such as a pyridine ring; or a        heteroatomic five-member ring, of the general form;    -   wherein, R¹⁴-R¹⁸ are independently a (—H) group; a hydroxyl        group (—OH); a methoxy group (—OCH₃); a nitro group (—NO₂), an        amine group (—NH₂), a halide; an alkoxy group having 1-20 carbon        atoms; an alkyl group having 1-20 carbon atoms; an aryl group        having 1-20 carbon atoms; an alkyl-amino group; an alkyl-thio        group; a cyano group (CN, SCN); an —CO₂H group; an —CO₂R group;        and the aromatic ring may be disubstituted, trisubstituted,        tetrasubstituted or pentasubstituted; and X is a —O, —N or —S,        or —SO, or —SO₂ group; and A is (CH₂)_(n) where n=0, 1, 2, 3, 4,        5, 6, 7, 8, 9 or 10; wherein if R⁷is a XAAR substituent R⁸ is        not and if R⁸ is a XAAR substituent R⁷is not.

Certain specific embodiments of the anthracyclines of the invention areshown in FIGS. 2-25.

The present application also comprises methods of preparing novelsubstituted sugar substrates and their use in the synthesis of the novelanthracycline analogs described in this invention. In certainembodiments the method for the synthesis of 4′-O-benzylated sugars isdescribed. These 4-O-benzylated sugars may comprise one of two mainclasses; glycal sugars or 1-O-silyalated sugars. Examples of thebenzylated sugars encompassed by this invention are WP567, WP735, WP736,WP819, W0820, WP821, WP822, WP823, WP824 and WP825.

Related embodiments describe the method for synthesizing glycals usingvarious bases including but not limited to NaH. Other relatedembodiments describe the synthesis of glycals using various solventsincluding but not limited to DMF. Yet other related embodiments describethe synthesis of glycals using various alkylating agents including butnot limited to benzyl chloride and benzyl bromide.

In certain embodiments, the method for the synthesis of amine containinganalogs of anthracyclines is described. Further embodiments describe theuse of substituted sugar azides for the synthesis of said aminecontaining anthracyclines wherein the azido substitution can be at the1′, 2′, 3′, 4′ or 5′ position on the sugar. The azide group serves as amasked or neutral form of amine substituent allowing for a couplingreaction (explained in Example 2, Procedure A). This allows thegeneration fo selective conditions to reduce azides (explained inExample 2, Procedure B). In one example the amine containinganthracyclines synthesized by this procedure are 14-OH analogs similarto doxorubicin, epirubicin or daunoribicin such as WP744 and WP769. Theazido sugar used in the preparation of this compound is WP819 (explainedin Example 1, Procedure A and B).

This procedure also allows the use of 14-O-blocked aglycons as theseblocked groups survive the steps in which azides are reduced and can beselectively removed at a later satge (Example 2, Procedure B).

The present application comprises methods of preparing substitutedanthracyclines and the preparation of important sugar substrates. Indevising the synthetic schemes and compounds of the present invention,the inventors have created a variety of novel compounds. These compoundsand their methods of synthesis are described elsewhere in thespecification, examples and figures and are given “WP” numbers. Thestructure of a compound designated with a “WP” number is ascertainableby reviewing the specification and figures. Exemplary specificanthracycline compounds that are encompassed by the invention are WP831,WP791, WP790, WP787, WP786, WP785, WP784, WP780, WP778, WP775, WP774,WP765, WP764, WP758, WP757, WP756, WP755, WP799, WP797, WP794, WP783,WP750, WP744, WP727 and WP571. Exemplary specific sugar substrates thatare encompassed by the invention are WP567, WP735, WP736, WP819, WP820,WP821, WP822, WP823, WP824, WP825.

The invention also considers methods of treating a patient with cancer,comprising administering to the patient a therapeutically effectiveamount of the contemplated substituted anthracycline compounds andtherapeutic kits comprising, in suitable container means, apharmaceutically acceptable composition comprising the contemplatedsubstituted anthracycline compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Structure of Doxorubicin and Daunorubicin

FIG. 2. Structure of WP831

FIG. 3. Structure of WP791

FIG. 4. Structure of WP790

FIG. 5. Structure of WP787

FIG. 6. Structure of WP786

FIG. 7. Structure of WP785

FIG. 8. Structure of WP784

FIG. 9. Structure of WP780

FIG. 10. Structure of WP778

FIG. 11. Structure of WP775

FIG. 12. Structure of WP774

FIG. 13. Structure of WP758

FIG. 14. Structure of WP757

FIG. 15. Structure of WP756

FIG. 16. Structure of WP755

FIG. 17. Structure of WP799

FIG. 18. Structure of WP797

FIG. 19. Structure of WP794

FIG. 20. Structure of WP783

FIG. 21. Structure of WP750

FIG. 22. Structure of WP744

FIG. 23. Structure of WP727

FIG. 24. Structure of WP567

FIG. 25. Structure of WP793

FIG. 26. Structure of WP819

FIG. 27. Structure of WP820

FIG. 28. Structure of WP821

FIG. 29. Structure of WP822

FIG. 30. Structure of WP823

FIG. 31. Structure of WP824

FIG. 32. Structure of WP825

FIG. 33. Structure of WP764

FIG. 34. Structure of WP765

FIG. 35. Synthesis of 4′-O-Benzylated Anthracyclines from 4-O-Benzylatedglycals

FIG. 36. Synthesis of 4′-O-Benzyl-3′-Amino-Anthracyclines

FIG. 37. Synthesis of 4-O-Benzyl-Glycals and its 3-O-Derivatives

FIG. 38. Synthesis of 4′-O-Benzyl-3′-Deamino-Anthracyclines from1-O-silylated 4-O-Benzyl-Hexopyranoses

FIG. 39. Examples of Selective Alkylation of Acylated Glycals

FIG. 40. Synthesis of 3′-Azido-Sugars and its 4-O-Benzylated Derivatives

FIG. 41. Alternative Synthesis of 3-Azido-4-O-Benzyl-Daunosamine from3-Azidoacosamine

FIG. 42. Structure of WP735

FIG. 43. Structure of WP736

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention provides new and novel DNA intercalating agents.These agents are substituted anthracyclines. These compounds show highactivity against resistant tumors and cells. A novel approach of theinvention produces compounds that are as active or more so than theparent compounds. Furthermore, the inventors' discovery is also for thedesign of effective DNA-binding substituted anthracyclines.

The anthracycline compounds have a tetracycline ring structure withsugars attached by a glycosidic linkage. Cytotoxic agents of this classhave quinone and hydroquinone moieties that permit them to function aselectron-accepting and electron donating agents. Doxorubicin anddaunorubicin are examples of compounds of this class (FIG. 1). Thesecompounds act by intercalating with DNA. Examples of exemplaryanthracyclinones and anthracyclines are given in Table 1. TABLE 1 Listof Exemplary anthracyclinones and anthracyclines. AnthracyclinonesRhodomycinone Isorhodomycinone Pyrromycinone 4-Demethoxydaunomycinone4-Demethoxyadriamycinone Daunomycinone Adriamycinone AnthracyclinesDaunorubicin Doxorubicin Epirubicin Idarubicin Pyrromycin AclacinamycineIsorhodomycine Carminomycine Doxorubicine 14-esters: Doxorubicin14-acetate Doxorubicin 14-propionate Doxorubicin 14-octanoateDoxorubicine 14-benzoate Doxorubicine 14-phenylacetate4′-Epidaunorubicin 4′-Epidoxorubicin 4′-Iododaunorubicin4′-Iododoxorubicin 4′-Deoxydaunorubicin 4′-Deoxydoxorubicin3′-Hydroxydaunorubicin 3′-Hydroxydoxorubicin 4-Demethoxydaunorubicin4-Demethoxydoxorubicin 4′-Epi-4-demethoxydaunorubicin4′-Epi-4-demethoxydoxorubicin

Following long-standing patent law convention, the words “a” and “an”,when used in the specification including the claims, denotes one ormore.

“Aryl” may be a phenyl or alkyl group, unsubstituted or substituted withan amine, alkylamine, nitro, carboxy, sulfonic acid, hydroxy, oxyalkyl,or halide.

The term “saccharide” includes oxidized, reduced or substitutedsaccharides. Saccharides of this invention include, but are not limitedto, ribose, arabinose, xylose, lyxose, allose, altrose, glucose,mannose, fructose, glucose, idose, galactose, talose, ribulose, sorbose,tagatose, gluconic acid, glucuronic acid, glucaric acididuronic acidrhamnose, fucose, N-acetyl glucosamine, N-acetyl galactosamine, N-acetylneuraminic acid, sialic acid, derivatives of saccharides such asacetals, amines, and phosphorylated sugars, oligosaccharides, as well asopen chain forms of various sugars, and the like.

An individual skilled in the art of organic synthesis in light of thepresent disclosure is able to prepare a large variety of substitutedsugars and substituted anthracyclines having C-3′-substituents or havingC4′-substitutents which are expected to have chemotherapeutic activitiesand may be used in the treatment of cancer and/or other diseases.Exemplary substituted anthracyclines having C-3′ alkylatedanthracyclines modified at benzyl ring of the present invention areWP831, WP791, WP790, WP787, WP786, WP785, WP784, WP780, WP778, WP775,WP774, WP758, WP757, WP756, WP755 (FIGS. 2-16). Exemplary substitutedanthracyclines having 4′-substituted-benzylated anthracyclines of thepresent invention are WP799, WP797, WP794, WP783, WP750, WP744, WP727and WP571 (FIGS. 17-25). Such specific anthracyclines having C-3′substituted moieties or having C-4′-substituted moieties have beensynthesized by the inventors and have been analyzed and the structureconfirmed by n.m.r and elemental analysis. The methods of the presentapplication enable one of skill in the art to synthesize these compoundsand many other related compounds without undue experimentation.

The present discoveries may be utilized in conjunction with certaintechniques that are well-known in the biological arts and that arefurther described in the following sections.

Pharmaceutical Compositions

The anti-tumor compounds of this invention can be administered to killtumor cells by any method that allows contact of the active ingredientwith the agent's site of action in the tumor. They can be administeredby any conventional methods available for use in conjunction withpharmaceuticals, either as individual therapeutically active ingredientsor in a combination of therapeutically active ingredients. They can beadministered alone but are generally administered with apharmaceutically acceptable carrier selected on the basis of the chosenroute of administration and standard pharmaceutical practice.

Aqueous compositions of the present invention will have an effectiveamount of anthracycline to kill or slow the growth of cancer cells.Further the potential recognition of genes can be accomplished by thesynthesis of substituted anthracyclines having C-3′ alkylatedanthracyclines modified at benzyl ring and having4′-substituted-benzylated anthracyclines with specific structures thatallow for the recognition of specific parts of DNA. Such compositionswill generally be dissolved or dispersed in a pharmaceuticallyacceptable carrier or aqueous medium.

The phrases “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, orhuman, as appropriate. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents for pharmaceuticalactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active ingredients,its use in the therapeutic compositions is contemplated. Supplementaryactive ingredients, such as other anti-cancer agents, can also beincorporated into the compositions.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms include, e.g., tablets or other solids for oraladministration; time release capsules; and any other form currentlyused, including cremes, lotions, mouthwashes, inhalants and the like.

A. Parenteral Administration

The active compounds will often be formulated for parenteraladministration, e.g., formulated for injection via the intravenous,intramuscular, subcutaneous, or even intraperitoneal routes. Thepreparation of an aqueous composition that contains an anthracycline ofthe present invention as an active ingredient will be known to those ofskill in the art in light of the present disclosure. Typically, suchcompositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for using to prepare solutions orsuspensions upon the addition of a liquid prior to injection can also beprepared; and the preparations can also be emulsified.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

In some forms, it will be desirable to formulate the novel compounds insalt form, generally to improve the solubility and bioavailability andto provide an active drug form more readily assimilated. As used herein,the term “pharmaceutically acceptable salt” refers to compounds whichare formed from acidifying a substituted anthracycline solution withsuitable physiologically tolerated acids. Suitable physiologicallytolerated acids are organic and inorganic acids, such as hydrochloricacid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalicacid, malonic acid, salicylic acid, maleic acid, methane sulfonic acid,isothionic acid, lactic acid, gluconic acid, glucuronic acid,amidosulfuric acid, benzoic acid, tartaric acid and pamoaic acid.Typically, such salt forms of the active compound will be provided ormixed prior to use.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

The active compounds may be formulated into a composition in a neutralor salt form. Pharmaceutically acceptable salts, include the acidaddition salts and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial ad antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

In certain cases, the therapeutic formulations of the invention couldalso be prepared in forms suitable for topical administration, such asin creams and lotions. These forms may be used for treatingskin-associated diseases, such as various sarcomas.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,with even drug release capsules and the like being employable.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will. be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 mL of isotonic NaCl solutionand either added to 1000 mL of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.

B. Oral Administration

In certain embodiments, active compounds may be administered orally.This is contemplated for agents which are generally resistant, or havebeen rendered resistant, to proteolysis by digestive enzymes. Suchcompounds are contemplated to include all those compounds, or drugs,that are available in tablet form from the manufacturer and derivativesand analogues thereof.

For oral administration, the active compounds may be administered, forexample, with an inert diluent or with an assimilable edible carrier, orthey may be enclosed in hard or soft shell gelatin capsule, orcompressed into tablets, or incorporated directly with the food of thediet. For oral therapeutic administration, the active compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparations should contain at least0.1% of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 2 to about 60% of the weight of the unit. The amount of activecompounds in such therapeutically useful compositions is such that asuitable dosage will be obtained.

The anthracycline analogs described in the present invention may beadministered alone or with cyclodextrins, or substituted cyclodextrinformulations.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, as gum tragacanth, acacia, cornstarch, or gelatin;excipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; and a sweetening agent, such as sucrose, lactoseor saccharin may be added or a flavoring agent, such as peppermint, oilof wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and propylparabens as preservatives, a dye and flavoring,such as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

Upon formulation, the compounds will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms, such as those described below in specificexamples.

Therapies

One of the major challenges in oncology today is the effective treatmentof a given tumor. Tumors are often resistant to traditional therapies.Thus, a great deal of effort is being directed at finding efficoustreatment of cancer. One way of achieving this is by combining new drugswith the traditional therapies and is discussed below. In the context ofthe present invention, it is contemplated that therapies using theanthracycline analogs could be used in conjunction with surgery,chemotherapy, radiotherapy and indeed gene therapeutic intervention. Italso may prove effective to combine the anthracycline analog basedchemotherapy with antisense or immunotherapies directed at tumor marker.

“Effective amounts” are those amounts of a candidate substance effectiveto reproducibly inhibit decrease, reduce, inhibit or otherwise abrogatethe growth of a cancer cell in an assay in comparison to levels inuntreated cells.

A. Standard Therapies

a. Chemotherapy:

A variety of chemical compounds, also described as “chemotherapeuticagents”, function to induce DNA damage, are used to treat tumors.Chemotherapeutic agents contemplated to be of use, include, adriamycin,5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D,mitomycin, cisplatin (CDDP), hydrogen peroxide, carboplatin,procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan,chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, tamoxifen, taxol, transplatinum,vincristin, vinblastin and methotrexate to mention a few.

Agents that damage DNA include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. A number of such agentshave been developed, particularly useful are agents that have undergoneextensive testing and are readily available. 5-fluorouracil (5-FU), isone such agent that is preferentially used by neoplastic tissue, makingit particularly useful for targeting neoplastic cells. Thus, althoughquite toxic, 5-FU, is applicable with a wide range of carriers,including topical and even intravenous administrations with dosesranging from 3 to 15 mg/kg/day.

Agents that directly cross-link nucleic acids, specifically DNA, areenvisaged to facilitate DNA damage leading to a useful antineoplastictreatment. For example, cisplatin, and other DNA alkylating agents maybe used. Cisplatin has been widely used to treat cancer, withefficacious doses used in clinical applications of 20 mg/m² for 5 daysevery three weeks for a total of three courses. Cisplatin is notabsorbed orally and must therefore be delivered via injectionintravenously, subcutaneously, intratumorally or intraperitoneally.

b. Radiotherapy:

Radiotherapeutic agents and factors include radiation and waves thatinduce DNA damage for example, γ-irradiation,X-rays, UV-irradiation,microwaves, electronic emissions, radioisotopes, and the like. Therapymay be achieved by irradiating the localized tumor site with the abovedescribed forms of radiations. It is most likely that all of thesefactors effect a broad range of damage DNA, on the precursors of DNA,the replication and repair of DNA, and the assembly and maintenance ofchromosomes.

Dosage ranges for X-rays range from daily doses of 50 to 200 roentgensfor prolonged periods of time (3 to 4 weeks), to single doses of 2000 to6000 roentgens. Dosage ranges for radioisotopes vary widely, and dependon the half-life of the isotope, the strength and type of radiationemitted, and the uptake by the neoplastic cells.

C. Surgery:

Surgical treatment for removal of the cancerous growth is generally astandard procedure for the treatment of tumors and cancers. Thisattempts to remove the entire cancerous growth. However, surgery isgenerally combined with chemotherapy and/or radiotherapy to ensure thedestruction of any remaining neoplastic or malignant cells.

B. Combination Therapies

To kill cells, inhibit cell growth, inhibit metastasis, inhibitangiogenesis or otherwise reverse or reduce the malignant phenotype oftumor cells, the methods of standard therapy discussed above aregenerally insufficient as tumors are often resistant to several of theseagents. Often combining a host of different treatment methods prove mosteffective in cancer therapy. Further, several AIDS afflicted patientshave a higher risk of developing cancers. Combination therapy in thesecases is required to treat AIDS as well as the cancer. Using the methodsand compounds developed in the present invention, one would generallycontact a “target” cell with an anthracycline analog synthesized in thepresent invention and at least one other agent. These compositions wouldbe provided in a combined amount effective to kill or inhibitproliferation of the cell. This process may involve contacting the cellswith the an anthracycline analog synthesized in the present inventionand the other agent(s) or factor(s) at the same time. This may also beachieved by contacting the cell with a single composition orpharmacological formulation that includes both agents, or by contactingthe cell with two distinct compositions or formulations, at the sametime, wherein one composition includes the anthracycline analogssynthesized herein and the other includes the agent.

Alternatively, the anthracycline analog-based treatment may precede orfollow the other agent treatment by intervals ranging from minutes toweeks. In embodiments where the other agent and anthracyclineanalog-based therapy are applied separately to the cell, one wouldgenerally ensure that a significant period of time did not expirebetween the time of each delivery, such that the agent and anthracyclineanalog-based treatment would still be able to exert an advantageouslycombined effect on the cell. In such instances, it is contemplated thatone would contact the cell with both modalities within about 12-24 h ofeach other and, more preferably, within about 6-12 h of each other, witha delay time of only about 12 h being most preferred. In somesituations, it may be desirable to extend the time period for treatmentsignificantly, however, where several days (2, 3, 4, 5, 6 or 7) toseveral weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

It also is conceivable that more than one administration of eitheranthracycline analog-based treatment or the other agent will be desired.Various combinations may be employed, where anthracycline analog-basedtreatment is “A” and the other agent is “B”, as exemplified below: A/B/AB/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/AB/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/BB/A/B/B B/B/A/B

Other combinations are also contemplated. Again, to achieve cellkilling, both agents are delivered to a cell in a combined amounteffective to kill the cell.

The invention also encompasses the use of a combination of one or moreDNA damaging agents, whether chemotherapeutic compounds orradiotherapeutics as described in the section above, together with theanthracycline analog. The invention also contemplates the use of theanthracycline analog in combination with surgical removal of tumors totreat any remaining neoplastic or metastasized cells. Further,immunotherapy may be directed at tumor antigen markers that are found onthe surface of tumor cells. The invention also contemplates the use ofC-3′ and C4′ substituted anthracycline analog-based treatments incombination with gene therapy, directed toward a variety of oncogenes,such as, tumor markers, cell cycle controlling genes. For example,combining the anthracycline analog-based treatment and gene therapytowards oncogenes such as p53, p16, p21, Rb, APC, DCC, NF-1, NF-2,BCRA2, pl6, FHIT, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc,neu, raf erb, src, fms, jun, trk ret, gsp, hst, bcl and abl mutations.

The other agent may be prepared and used as a combined therapeuticcomposition, or kit, by combining it with the anthracycline analog-basedtreatment, as described above. The skilled artisan is directed to“Remington's Pharmaceutical Sciences” 15th Edition, chapter 33, inparticular pages 624-652. Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

It is proposed that the regional delivery of anthracycline analog-baseddrugs described in the present invention to patients with tumors will bea very efficient method for delivering a therapeutically effectivechemical to counteract the clinical disease. Similarly, otherchemotherapeutics, radiotherapeutics, gene therapeutic agents may bedirected to a particular, affected region of the subjects body.Alternatively, systemic delivery of anthracycline analog-based treatmentand/or the agent may be appropriate in certain circumstances, forexample, where extensive metastasis has occurred.

It also should be pointed out that any of the standard or othertherapies may prove useful by themselves in treating a cancer. In thisregard, reference to chemotherapeutics and anthracycline analog-basedtreatment in combination should also be read as a contemplation thatthese approaches may be employed separately.

When such combination therapy is employed for the treatment of a tumor,the cytotoxic agent may be administered at a dosage known in the art tobe effective for treating the tumor. However, the anthracyclineanalog-based compounds may produce an additive or synergistic effectwith a cytotoxic agent against a particular tumor. Thus, when suchcombination therapy is used, the dosage of anthracycline analog-baseddrugs administered may be less than that administered when the cytotoxicagent is used alone. Similarly, for patients afflicted by AIDS,AZT/protease inhibitors will be used with anthracycline analogcompounds, or other herein mentioned therapeutic agent(s). Again thedosage of anthracycline analog-based compounds or other conjunctivelyutilized agent, may be altered to suit the AIDS treatment.

Preferably, the patient is treated with anthracycline analog-basedcompounds for about 1 to 14 days, preferably 4 to 14 days, prior to thebeginning of therapy with a cytotoxic agent, and thereafter, on a dailybasis during the course of such therapy. Daily treatment with theanthracycline analogs can be continued for a period of, for example, 1to 365 days after the last dose of the cytotoxic agent is administered.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 Synthesis of Exemplary Substituted Anthracyclines having C-3′Alkylated Anthracyclines Modified at Benzyl Ring

General Procedure for Making C-3′ Alkylated Anthracyclines Modified atBenzyl Ring

A. Synthesis of 3′-N-nonitro benzyl Derivatives of Daunorubicin and4-demethoxy-daunorubicin:

1. Procedure A

Daunorubicin (1 mmol), 4-nitrobenzyl bromide (1 mmol), and sodiumcarbonate (250 g) were dissolved in DMF (10 ml). Dichloromethane (10 ml)was added to this solution, and obtained mixture was stirred at roomtemperature until all substrate was converted into the product.(monitored by TLC). After reaction was completed the reaction mixturewas diluted with dichloromethane (100 ml), and washed with water untilneutral. The organic solution was dried over sodium sulfate, then dryingagent was removed and solvent evaporated under diminished pressure, andproduct was purified by column chromatography (Silica Gel 60 Merck),using chloroform, chloroform/methanol 98:2, 95:5 as an eluent.

1.1 The Following Compounds were Obtained from Daunorubicin Accordingthe Above Procedure (Procedure A):

(i) WP 756 FIG. 15

¹H-n.m.r (CDCl₃) δ: 13.99, 13.40 (2s, 1H ea, 6,11-OH), 8.05 (d, 1H,J=7.5 Hz, H-1), 7.93 (d, 1H, J=8.1 Hz, H-2), 7.80 (dd, 1H, J=8.2 Hz,H-3), 7.60-7.52 (m, 2H, H-aromatic), 7.44-7.40 (m, 2H, H-aromatic), 5.54(d, J=3.2 Hz, H-1′), 5.32 (, 1H, H-7), 4.67 (s, 1H, 9-OH), 4.13-4.06 (m,4H, OMe and H-5′), 4.04 (d, 1H, J=13.6 Hz, Ch2Ph), 3.95 (d, 1H, J=13.6Hz, Ch2Ph), 3.72 (, 1H, H-4′), 3.25 (dd, 1H, J=18.1 Hz, J=1.1 Hz, H=10),2.97 (d, 1H, J=18.9 Hz, H-10), 3.00-2.92 (m, 1H, H-3′), 2.44 (s, 3H,14-CH₃), 2.38 (d, 1H, J=14.82 Hz, H-8), 2.12 (dd, 1H, J=14.8 Hz, J=4.0Hz, H-8), 1.86-1.69 (m, 2H, H-2′a,e), 1.4 (d, 3H, J=6.6 Hz, H-6′). Anal.Elem. Calcd. for: C₃₄H₃₅O₁₂N₂Cl×1.5 H₂O: C: 56.24; H: 5.27; N: 3.86; Cl:4.88. Found: C: 56.24; H: 5.27; N: 3.86; Cl: 4.88.

(ii) WP 784 FIG. 8

¹H-n.m.r (CDCl₃) δ: 13.98, 13.29 (2s, 1H ea, 6, 11-OH), 8.14 (s, 1H,H-aromatic), 8.08 (d, 1H, J=8.1 Hz, H-arom), 8.03 (d, 1H, J=7.6 Hz,H-1), 7.78 (dd, 1H, J=J=8.15 Hz H-2), 7.62 (d, 1H, J=7.6 Hz,H-aromatic), 7.46 (dd, 1H, J=J=7.9 Hz, H-aromatic), 7.39 (d, 1H, J=8.4Hz, H-aromatic) 5.53 (d, J=3.0 Hz, H-1′), 5.3 (, 1H, H-7), 4.62 (s, 1H,9-OH), 4.10-4.08 (m, 1H, H-5′), 4.08 (s, 3H, OMe), 3.9 (d, 1H, J=13.6Hz, Ch2Ph), 3.81 (d, 1H, J=13.6 Hz, Ch2Ph), 3.67 (, 1H, H-4′), 3.22 (dd,1H, J=18.8 Hz, J=1.8 Hz, H=10), 2.96 (d, 1H, J=18.8 Hz, H-10), 2.96-2.92(m, 1H, H-3′), 2.41 (s, 3 H, 14-CH₃), 2.36 (d, 1H, J=14.8 Hz, H-8), 2.11(dd, 1H, J=14.8Hz, J=2.1 Hz, H-8), 1.82-1.75 (m, 2H, H-2′a,e), 1.37 (d,3H, J=6.5 Hz, H-6′). Anal. Elem. Calcd. for: C₃₄H₃₅O₁₂N₂Cl×1.5 H₂O: C:56.24; H: 5.27; N: 3.86; Cl: 4.88. Found: C: 55.94; H: 5.27; N: 3.78;Cl: 4.92.

(iii) WP 755 FIG. 16

¹H-n.m.r (CDCl₃) δ: 14.02, 13.30 (2s, 1H ea, 6,11-OH), 8.17 (d, 1H,J=8.6 Hz, H-aromatic), 8.05 (d, 1H, J=7.8 Hz, H-1), 7.81 (dd, 1H,J=J=7.8 Hz, H-2), 7.49-7.41 (m, 4H, H-3 and H-aromatic), 5.54 (s, 1H,H-1′), 5.32 (, 1H, H-7), 4.64 (s, 1H, 9-OH), 4.11 (s, 3H, OMe), 4.09 (q,1H, J=6.5 Hz, H-5′), 3.93 (d, 1H, J=14 Hz, Ch2Ph), 3.83 (d, 1H, J=14 Hz,Ch2Ph), 3.67 (, 1H, H-4′), 3.25 (d, 1H, J=18.9 Hz, H=10), 2.98 (d, 1H,J=18.9 Hz, H-10), 2.96-2.92 (m, 1H, H-3′), 2.43 (s, 3H, 14-CH₃), 2.38(d, 1H, J=15.2 Hz, H-8), 2.13 (dd, 1H, J=15.2 Hz, J=4.13 Hz, H-8),1.82-1.78 (m, 2H, H-2′a,e), 1.38 (d, 3H, J=6.6 Hz, H-6′). Anal. Elem.Calcd. for: C₃₄H₃₅O₁₂N₂Cl×H₂O: C: 56.95; H: 5.2; N: 3.91; Cl: 4.94.Found: C: 56.99; H: 5.19; N: 3.81; Cl: 4.89.

1.2 The following compounds were obtained from 4-demethoxy-daunorubicinusing Procedure A:

(iv) WP 790 FIG. 4

¹H-n.m.r (CDCl₃) δ: 13.58, 13.34 (2s, 1H ea, 6,11-OH), 8.36-8.32 (m, 2H,H-1,4), 8.90 (d, 1H, J=8.2 Hz, H-arom), 7.85-7.80 (m, 2H, H-2,3), 7.57(dd, 1H, J=J=7.5 Hz, H-aromatic), 7.51 (d, 1H, J=6.5 Hz, H-aromatic),7.40 (dd, 1H, J=J=7.1 Hz, H-aromatic), 5.50 (d, 1H, J=3.6 Hz, H-1′),5.28 (, 1H, H-7), 4.66 (s, 1H, 9-OH), 4.10 (m, 1H, J=6.6 Hz, H-5′), 4.03(d, 1H, J=14.7 Hz, Ch2Ph), 3.94 (d, 1H, J=14.7 Hz, Ch2Ph), 3.70 (, 1H,H-4′), 3.25 (dd, 1H, J=19 Hz, J=1.7 Hz, H=10), 3.00 (d, 1H, J=19 Hz,H-10), 2.96-2.91 (m, 1H, H-3′), 2.40 (s, 3H, 14-CH₃), 2.38 (d, 1H,J=14.9 Hz, H-8), 2.10 (dd, 1H, J=14.9 Hz, J=4.1 Hz, H-8), 1.82 (ddd, 1H,J=13.3 Hz, J=4.2 Hz, H-2′a), 1.74 (dd, 1H, J=13.3 Hz, J=4.9 Hz, H-2′e),1.38 (d, 3H, J=6.6 Hz, H-6′).

Anal. Elem. Calcd. for: C₃₃H₃₃O₁₁N₂Cl×1.5 H₂O: C: 56.94; H: 5.21; N:4.02; Cl: 5.09. Found: C: 57.00; H: 5.13; N: 3.98; Cl: 5.16.

(v) WP 774 FIG. 12

¹H-n.m.r (CDCl₃) δ: 13.59, 13.35 (2s, 1H ea, 6,11-OH), 8.37-8.32 (m, 2H,H-1,4), 8.13 (d, 1H, J=8.2 Hz, H-arom), 7.86-7.82 (m, 2H, H-2,3), 7.44(d, 2H, H-aromatic), 5.50 (s, 1H, H-1′), 5.28 (, 1H, H-7), 4.62 (s, 1H,9-OH), 4.09 (m, 1H, J=6.4 Hz, H-5′), 3.91 (d, 1H, J=14.7 Hz, Ch2Ph),3.83 (d, 1H, J=14.7 Hz, Ch2Ph), 3.66 (, 1H, H-4′), 3.25 (dd, 1H, J=19Hz, J=1.8 Hz, H=10), 2.98 (d, 1H, J=19 Hz, H-10), 2.96-2.93 (m, 1H,H-3′), 2.40 (s, 3H, 14-CH₃), 2.37 (d, 1H, J=13.0 Hz, H-8), 2.11 (dd, 1H,J=13.0 Hz, J=4.0 Hz, H-8), 1.84-1.76 (m, 2H, H-2′a,e), 1.28 (d, 3H,J=6.4 Hz, H-6′). Anal. Elem. Calcd. for: C₃₃H₃₃O₁₁N₂Cl×0.5 H₂O: C:57.15; H: 4.87; N: 4.04; Cl: 5.11. Found: C: 57.15; H: 5.08; N: 4.01;Cl: 5.11.

1.3 The Following Compound WP787 was Obtained when 4-amino-daunorubicinUsing benzyl bromide According to Procedure A:

(vi) WP 787 FIG. 5

¹H-n.m.r (CDCl₃) δ: 13.98, 13.29 (2s, 1H ea, 6,11-OH), 8.02 (d, 1H,J=7.3 Hz, H-1), 7.81 (dd, 1H, J=7.8 Hz, H-2), 7.40 (d, 1H, J=8.1 Hz,H-3), 7.35 (s, 4H, H-aromatic), 7.31-7.26 (m, 1H, H-aromatic), 5.42 (d,1H, J=4.0 Hz, H-1′), 5.25 (, 1H, H-7), 4.64 (s, 1H, 9-OH), 4.14 (q, 1H,J=6.6 Hz, H-5′), 4.11 (d, 1H, J=12.7 Hz, Ch2Ph), 4.07 (s, 3H, OMe), 3.77(d, 1H, J=12.7 Hz, Ch2Ph), 3.72-3.68 (m, 1H, H-3′), 3.22 (dd, 1H, J=18.8Hz, J=1.8 Hz, H=10), 2.94 (d, 1H, J=18.8 Hz, H-10), 2.76 (d, 1H, J=3.9Hz, H-4′), 2.41 (s, 3H, 14-CH₃), 2.29 (d, 1H, J=14.7 Hz, H-8). Anal.Elem. Calcd. for: C₃₄H₃₆O₁₀NCl: C: 62.43; H: 5.55; N: 2.14; Cl: 5.42.

(vii) WP 831 FIG. 2 The Following Compound was Obtained fromdaunorubicin Using 4-picolyl chloride According to Procedure A:

¹H-n.m.r (CDCl₃) δ: 13.97, 13.37 (2s, 1H ea, 6,11-OH), 8.50 (d, 2H,H-arom), 8.02 (d, 1H, J=7.5 Hz, H-1), 7.78 (dd, 1H, J=7.8 Hz, H-2), 7.39(d, 1H, H-3), 7.18 (m, 2H, H-aromatic), 5.51 (d, 1H, J=3.8 Hz, H-1′),5.28 (, 1H, H-7), 4.62 (s, 1H, 9-OH), 4.08 (m, 4H, H-5′ and OMe), 3.81(d, 1H, J=14.2 Hz, Ch2Ph), 3.72 (d, 1H, J=14.2 Hz, Ch2Ph), 3.64 (, 1H,H-4′), 3.37 (dd, 1H, J=19 Hz, J=1.5 Hz, H=10), 2.94 (d, 1H, J=19 Hz,H-10), 2.96-2.93 (m, 1H, H-3′), 2.41 (s, 3H, 14-CH₃), 2.36 (d, 1H,J=14.8 Hz, H-8), 2.10 (dd, 1H, J=14.8 Hz, J=4.1 Hz, H-8), 1.83-1.75 (m,2H, H-2′a,e), 1.36 (d, 3H, J=6.4 Hz, H-6′). Anal. Elem. Calcd. for:C₃₃H₃₆O₁₀N₂Cl₂×3 H₂O: C: 53.16; H: 5.68; N: 3.76; Cl: 9.51. Found: C:53.70; H: 5.59; N: 3.67; Cl: 9.36.

1.4 The Following Compound WP 786 was Obtained according Procedure AUsing 4-fluorobenzyl bromide as alkylating Agent:

(viii) WP 786 FIG. 6

¹H-n.m.r (CDCl₃) δ: 13.98, 13.30 (2s, 1H ea, 6,11-OH), 8.03 (d, 1H,J=7.6 Hz, H-1), 7.78 (dd, 1H, J=7.6 Hz, H-2), 7.39 (d, 1H, J=7.8 Hz,H-3), 7.23-7.20 (m, 2H, H-aromatic), 7.00-6.95 (m, 2H, H-aromatic), 5.51(d, 1H, J=3.7 Hz, H-1′), 5.3 (, 1H, H-7), 4.69 (s, 1H, 9-OH), 4.08 (s,3H, OMe), 4.06 (q, 1H, J=6.6 Hz, H-5′), 3.96 (d, 1H, J=12.7 Hz, Ch2Ph),3.64 (d, 1H, J=12.7 Hz, Ch2Ph), 3.64 (, 1H, H-4′), 3.23 (dd, 1H, J=18.9Hz, J=1.9 Hz, H=10), 2.96 (d, 1H, J=18.9 Hz, H-10), 2.96-2.93 (m, 1H,H-3′), 2.42 (s, 3H, 14-CH₃), 2.37 (d, 1H, J=13.8 Hz, H-8), 2.09 (dd, 1H,J=13.8 Hz, J=4.1 Hz, H-8), 1.78 (ddd, 1H, J=13.1 Hz, J=4.0 Hz, H-2′a),1.67 (dd, 1H, J=13.1 Hz, J=4.9 Hz, H-2′e), 1.37 (d, 3H, J=6.6 Hz, H-6′).

B. Synthesis of 3′-N-nitro benzyl Derivatives of doxorubicin and4-demethoxydoxorubicin:

2. Procedure B

Doxorubicin having hydroxyl at C-14 protected by silylation (1 mmol),4-nitrobenzyl bromide (1 mmol), and sodium carbonate (250 mg) weredissolved in DMF (10 ml). Dichloromethane (10 ml) was added to thissolution, and obtained mixture was stirred at room temperature until allsubstrate was converted into the product. (monitored by TLC control).After reaction was completed the reaction mixture was diluted withdichloromethane (100 ml), and washed with water until neutral. Theorganic solution was dried over sodium sulfate, then drying agent andsolvent were removed, and product was purified by column chromatography(Silica Gel 60 Merck), using chloroform, chloroform/methanol 98:2, aseluent.

Obtained compounds was dissolved in THF (6 ml) and to this 1 N watersolution of HCl (9 ml) was added. The mixture was stirred at room temp.overnight, then it was diluted with chloroform, and neutralized with 10%water solution of potassium bicarbonate. Organic solution was dried oversodium sulfate. Drying agent was filtered off, and solvent wasevaporated to dryness. Crude product was purified by columnchromatography (Silica Gel 60 Merck), using chloroform/methanol 98:2,95:5 as eluent.

The Following Compounds were Obtained according to this Procedure B:

(ix) WP 780 FIG. 9

¹H-n.m.r (CDCl₃) δ: 13.96, 13.26 (2s, 1H ea, 6,11-OH), 8.03 (d, 1H,J=7.7 Hz, H-aromatic), 7.91 (d, 1H, J=8.1 Hz, H-1), 7.79 (dd, 1H, J=8.1Hz, H-2), 7.56 (dd, 1H, J=J=7.8 Hz H-aromatic), 7.50 (d, 1H, J=6.8 Hz,H-aromatic), 7.40-7.30 (m, 2H, H-3 and H-aromatic), 5.52 (d, 1H, J=3.6Hz, H-1′), 5.3 (, 1H, H-7), 4.77 (s, 2H, 14-CH₂), 4.75 (s, 1H, 9-OH),4.08 (s, 3H, OMe), 4.03 (d, 1H, J=13.6 Hz, Ch2Ph), 3.99 (q, 1H, J=6.6Hz, H-5′) 3.93 (d, 1H, J=13.6 Hz, Ch2Ph), 3.70 (, 1H, H-4′), 3.27 (dd,1H, J=18.9 Hz, J=1.7 Hz, H=10), 3.03 (d, 1H, J=18.9 Hz, H-10), 2.89 (m,1H, H-3′), 2.36 (d, 1H, J=14.8 Hz, H-8), 2.16 (dd, 1H, J=14.8 Hz, J=2.9Hz, H-8), 1.84 (ddd, 1H, J=13.1 Hz, J=4.0 Hz, H-2′a), 1.68 (dd, 1H,J=13.1 Hz, J=4.6 Hz, H-2′e), 1.39 (d, 3H, J=6.6 Hz, H-6′). Anal. Elem.Calcd. for: C₃₄H₃₅O₁₃N₂Cl×2 H₂O: C: 54.37; H: 5.23; N: 3.73; Cl: 4.72.Found: C: 54.49; H: 5.13; N: 3.70; Cl: 4.76.

(x) WP 765 FIG. 34

¹H-n.m.r (CDCl₃) δ: 13.96, 13.24 (2s, 1H ea, 6,11-OH), 8.13 (d, 1H,J=7.6 Hz, H-1), 8.01 (d, 1H, J=7.5 Hz, H-2), 7.71 (d, 1H, J=7.8 Hz,H-3), 7.45-7.40 (m, 4H, H-aromatic), 5.52 (, 1H, H-1′), 5.31 (, 1H,H-7), 4.75 (s, 2H, 14-CH₂), 4.70 (s, 1H, 9-OH), 4.00 (s, 3H, OMe), 3.99(q, 1H, J=6.6 Hz, H-5′) 3.91 (d, 1H, J=14.0 Hz, Ch2Ph), 3.82 (d, 1H,J=14.0 Hz, Ch2Ph), 3.66 (, 1H, H-4′), 3.24 (d, 1H, J=18.8 Hz, H=10),3.00 (d, 1H, J=18.8 Hz, H-10), 2.90-2.88 (m, 1H, H-3′), 2.37 (d, 1H,J=14.8 Hz, H-8), 2.15 (dd, 1H, J=14.8 Hz, J=4.0 Hz, H-8), 1.8-1.6 (m,2H, H-2′a,e), 1.39 (d, 3H, J=6.5 Hz, H-6′). Anal. Elem. Calcd. for:C₃₄H₃₅O₁₃N₂Cl×0.5 H₂O: C: 59.39; H: 5.13; N: 4.07. Found: C: 59.00; H:5.15; N: 4.07

2.1 The Follwing Compound WP778 was Obtained from 14-O-silylated4-demethoxydoxorubicin Following Compound was Prepared AccordingProcedure B:

(xi) WP 778 FIG. 10

¹H-n.m.r (CDCl₃) δ: 13.57, 13.30 (2s, 1H ea, 6,11-OH), 8.36-8.32 (m, 2H,H-1,4), 8.13 (d, 2H, J=8.7 Hz, H-arom), 7.88-7.83 (m, 2H, H-2,3), 7.44(d, 2H, J=8.7 Hz, H-aromatic), 5.52 (d, 1H, J=3.3 Hz, H-1′), 5.30 (, 1H,H-7), 4.76 (, 2H, 14-CH₂), 4.73 (s, 1H, 9-OH), 4.01 (m, 1H, J6.6 Hz,H-5′), 3.93 (d, 1H, J=14.1 Hz, Ch2Ph), 3.83 (d, 1H, J=14.1 Hz, Ch2Ph),3.68 (, 1H, H-1′), 3.29 (dd, 1H, J=19 Hz, J=1.8 Hz, H=10), 3.06 (d, 1H,J=19 Hz, H-10), 2.96-2.88 (m, 1H, H-3′), 2.38 (d, 1H, J=14.7 Hz, H-8),2.15 (dd, 1H, J=14.7 Hz, J=4.0 Hz, H-8), 1.88-1.74 (m, 1H, H-2′a,e),1.29 (d, 3H, J=6.6 Hz, H-6′).

C. Synthesis of 3′-N-amino-substituted benzyl Derivatives ofdaunorubicin and 4-methoxy-daunorubicin:

3. Procedure C

Products obtained according to the procedure A and B (0.1 mmol) weredissolved in the mixture of dichloromethane and methyl alcohol (1: Iv/v) (10 ml), then stannous chloride (II) (1.1 g) was added and themixture was stirred at room temp. until all substrate disappeared (TLCcontrol). The reaction mixture was then diluted with chloroform, andsaturated solution of sodium bicarbonate (100 ml) was added. Thereaction mixture was stirred at room temp. for 3 hr, then the inorganicsalts were filtered off, and washed with chloroform. Organic solutionwas washed with water until neutral, then dried over Na₂SO₄. Dryingagent and solvent were removed, and product was purified by columnchromatography (Silica Gel 60 Merck), using chloroform/methanol 98:2,95:5, 9:1 as an eluent.

Following compounds were prepared according to the Procedure C:

(xii) WP 758 FIG. 13

¹H-n.m.r (CDCl₃) δ: 13.98, 13.28 (2s, 1H ea, 6,11-OH), 8.04 (d, 1H,J=8.0 Hz, H-1), 7.78 (dd, 1H, J=8.0 Hz, H-2), 7.38 (d, 1H, J=8.0 Hz,H-3), 7.05 (td, 1H, J=7.6 Hz, J=1.3 Hz, H-aromatic), 6.97 (dd, 2H, J=7.8Hz, J=1.4 Hz, H-aromatic), 6.63 (m, 2H, H-aromatic), 5.51 (, 1H, H-1′),5.28 (, 1H, H-7), 4.08 (s, 3H, OMe), 4.13-4.08 (q, 1H, J=6.5 Hz, H-5′),3.82 (d, 1H, J=12.5 Hz, Ch2Ph), 3.73 (, 1H, H-4′), 3.71 (d, 1H, J=12.5Hz, Ch2Ph), 3.23 (dd, 1H, J=18.9 Hz, J=1.7 Hz, H=10), 2.95 (d, 1H,J=18.9 Hz, H-10), 2.99-2.93 (m, 1H, H-3′), 2.44 (s, 3H, 14-CH₃), 2.7 (,2H, NH, OH), 2.34 (d, 1H, J=14.9 Hz, H-8), 2.10 (dd, 1H, J=14.9 Hz,J=4.1 Hz, H-8), 1.81-1.74 (m, 2H, H-2′a,e), 1.37 (d, 3H, J=6.6 Hz,H-6′). Anal. Elem. Calcd. for: C₃₄H₃₈O₁₀N₂Cl₂: C: 57.88; H: 5.43;N:3.97; Cl: 10.05. Found: C: 57.85; H: 5.44; N: 3.93; Cl: 9.96

(xiii) WP 757 FIG. 14

¹H-n.m.r (CDCl₃) δ: 14.00 (, 1H, H-6 or H-11), 8.06 (d, 1H, J=7.7 Hz,H-1), 7.80 (dd, 1H, J=8.1 Hz, H-2), 7.41 (d, 1H, J=7.7 Hz, H-3), 7.05(d, 2H, J=8.3 Hz, H-aromatic), 6.62 (d, 2H, J=8.3 Hz, H-aromatic), 5.53(d, 1H, J=3.4 Hz, H-1′), 5.31 (, 1H, H-7), 4.75 (s, 1H, 9-OH), 4.10 (s,3H, OMe), 4.10 (q, 1H, J=6.5 Hz, H-5), 3.71 (d, 1H, J=15.0 Hz, Ch2Ph),3.7 (, 1H, H-4′), 3.58 (d, 1H, J=15.0 Hz, Ch2Ph), 3.28 (dd, 1H, J=19.0Hz, J=1.0 Hz, H=10), 3.0 (d, 1H, J=19.0 Hz, H-10), 3.02-2.92 (m, 1H,H-3′), 2.45 (s, 3H, 14-CH₃), 2.40 (d, 1H, J=14.9 Hz, H-8), 2.11 (dd, 1H,J=14.4 Hz, J=4.0 Hz, H-8), 1.78 (ddd, 1H, J=12.5 Hz, J=8.6 Hz, J=3.8 Hz,H-2′a), 1.63 (dd, 1H, J=12.5 Hz, J=5.2 Hz, H-2′e) 1.40 (d, 3H, J=6.5 Hz,H-6′). Anal. Elem. Calcd. for: C₃₄H₃₆O₁₀N₃: C: 64.55; H: 5.74; N: 4.43.Found: C: 64.27; H: 5.90; N: 4.28

(xiv) WP 791 FIG. 3

¹H-n.m.r (CDCl₃) δ: 8.37 (m, 2H, H-1,4), 7.87-7.83 (m, 2H, H-2,3), 7.04(dd, 1H, J=8.0 Hz, H-aromatic), 6.97 (d, 2H, J=7.1 Hz, H-aromatic),6.66-6.62 (m, 2H, H-aromatic), 5.51 (d, 1H, J=3.5 Hz, H-1′), 5.29 (, 1H,H-7), 4.67 (s, 1H, 9-OH), 4.11 (q, 1H, J=6.5 Hz, H-5′), 3.80 (d, 1H,J=12.3 Hz, Ch2Ph), 3.7 (, 1H, H-4′), 3.68 (d, 1H, J=12.3 Hz, Ch2Ph),3.27 (dd, 1H, J=19.0 Hz, J=1.4 Hz, H=10), 3.0 (d, 1H, J=19.0 Hz, H-10),2.97-2.88 (m, 1H, H-3′), 2.40 (s, 3H, 14-CH₃), 2.37 (d, 1H, J=15.2 Hz,H-8), 2.11 (dd, 1H, J=15.2 Hz, J=4.0 Hz, H-8), 1.89-1.73 (m, 2H,H-2′a,e), 1.35 (d, 3H, J=6.5 Hz, H-6′).

(xv) WP 764 FIG. 33

¹H-n.m.r (DMSO-d6) δ: 7.91-7.89 (m, 2H, H-1,2), 7.62-7.60 (m, 1H, H-3),7.00 (d, 2H, J=8.2 Hz, H-arom), 6.5 (d, 2H, J=8.2 Hz, H-aromatic), 5.19(d, 1H, J=3.4 Hz, H-1′), 4.89 (, 1H, H-7), 4.38 (s, 1H, 9-OH), 4.14 (q,1H, J=6.5 Hz, H-5′), 3.98 (s, 3H, OMe), 3.81 (m, 1H, H-4′), 3.78 (s, 2H,H-aromatic), 2.96 (d, 1H, J=18.0 Hz, H=10), 2.88 (d, 1H, J=19.0 Hz,H-10), 2.57 (m, 1H, H-3′), 2.25 (s, 3H, 14-CH₃), 2.14 (dd, 1H, J=14.3Hz, J=3.0 Hz, H-8), 2.07 (dd, 1H, J=14.3 Hz, J=5.0 Hz, H-8), 1.66 (ddd,1H, J=14.2 Hz, J=13.0 Hz, J=3.5 Hz, H-2′a), 1.55 (dd, 1H, J=13.0 Hz,J=4.4 Hz, H-2′e) 1.15 (d, 3H, J=6.5 Hz, H-6′).

EXAMPLE 2

Synthesis of Exemplary C-4′-Alkyl-Aromatic Ring Anthracyclines

General Procedures for Making Anthracycline Analogs withC-4′-substituted Sugars.

A. Synthesis of 4′-O-benzylated anthracyclines

1. Procedure A: Coupling and Deacetylation.

In the one flask a mixture of aglycon (1 mmol), HgBr₂ (0.7 g), HgO (2.8g) and molecular sieves 4 A° (1 g) in dichloromethane (50 ml) wasprepared and stirred at room temperature. At the same time, in thesecond flask, a trimethylsilyl bromide (3 mmol) was added to thesolution of 1-O-silylated 3-azido-sugar (2 mmol) in dichloromethane (8mL). Such reaction mixture was stirred at RT and its progress wasmonitored by TLC. (Toluene/acetone=8:1). After disappearance of startingsugar, solvent was removed under diminished pressure. Then hexanes (10mL) were added to the residue and the mixture was evaporated. Suchaddition and removal of hexanes were repeated three times. Subsequently,the resulting dry residue was dissolved in the dichloromethane (10 mL).This solution was added in three portions to the first flask containingearlier prepared solution of aglycone and mercuric salts. The finalreaction mixture was stirred at RT for 10 min. (progress of the reactionwas monitored by TLC), then it was diluted with dichloromethane (50 mL),and filtered through Celite. Filtrate was washed with 10% water soln. ofKI, then twice with water, and it was dried over Na₂SO₄. The crudeproduct was purified by column chromatography and subsequentlydeacetylated in the mixture of methylene chloride and methanol (2:1,v/v) (50 mL), using solid potassium carbonate (2 g). The reactionmixture was stirred at RT until all substrate disappeared. (progress ofthe reaction was monitored by TLC). The mixture was diluted withdichloromethane (100 mL), then 1N water solution of HCl (15 mL), andwater (50 mL) were added. The layers were separated. The organic layerwas washed with water until neutral, then with brine, and it was driedover Na₂SO₄. The product was purified by column chromatography.

2. Procedure B: Reduction and Desilylation.

The coupling product containing azido group at the C-3′ position (1mmol) was dissolved in THF (8 mL). Triphenyl phosphine (2 mmol) wasadded to this solution, and reaction mixture was stirred at roomtemperature overnight. Progress of the reaction was monitored by TLCuntil disappearance of substrate. Then the reaction mixture was dilutedwith chloroform (50 mL) and 2N NH₃ in methanol (2 mL) was added to thissolution. Reaction mixture was stirred for 1 h, then the solution waswashed with water until neutral, then with brine, and dried over Na₄SO₄.Obtained crude product was purified by column chromatography.Anthracycline antibiotic with hydroxyl protected at 14 (1 mmol) wasdissolved in THF (10 mL), then 1N HCl (15 mL) was added and the mixturewas stirred at room temperature until all substrate disappeared as judgeby TLC. The mixture was diluted with chloroform (20 mL). The layers wereseparated and the water solution was washed with chloroform untilorganic layer was colorless, and then saturated solution of NaHCO₃ wasadded to adjust pH to 8-9. Basic solution was extracted with chloroformcontained 5% of methanol. Combined extracts were washed with brine anddried over dry Na₂SO₄. The solvent was removed under diminished pressureand dry product was dissolved in methanol (4 mL). 1N solution of HCl inmethanol (1.5 mL) followed by diethyl ether were added to this solution.Obtained solid hydrochloride was filtered off and washed with diethylether to remove excess of acid. When the filtrate was neutral, solidhydrochloride was precipitated from methanol/diethyl ether to giveanalytically pure product.

(i) WP 750A FIG. 36

3′-Deamino-3′-azido-4′-O-benzyl-14-O-tert-butyldiphenylsilyl-4′-epidoxorubicin

Product was obtained according to the A. Yield 85%.

nmr (CDCl₃) δ: 14.00, 13.19 (2s, 1H each, 6-OH, 11-OH), 8.02 (dd, 1H,J=8.47 Hz, J=0.98 Hz, H-1), 7.77 (dd, 1H, J=J=8.39 Hz, H-2), 7.70-7.76(m, 4H), 7.45-7.30 (m, 12H, H-3), 5.39 (d, IH, J=3.72 Hz, H-1′), 5.17(bs, 1H, H-7), 4.89 (s, 2H, 14-CH₂), 4.81 (d, 1H, J=10.60 Hz, CH₂Ph),4.60 (d, 1H, J=10.60 Hz, CH₂Ph), 4.16 (s, 1H, 9-OH), 4.07 (s, 3H, OMe),3.71 (dq, 1H, J=9.4 Hz, J=6.2Hz, H-5′), 3.56 (ddd, 1H, J=12.53 Hz,J=9.38 Hz, J=4.91 Hz, H-3′), 2.97 (dd, 1H, J=J=9.4 Hz, H-4′), 2.97 (dd,1H, J=18.94 Hz, J=1.24 Hz, H-10), 2.76 (d, 1H, J=18.94 Hz, H-10),2.1-2.0 (m, 3H, H-8a,e, and H-2′e), 1.64 (ddd, 1H, J=13.28 Hz, J=13.28Hz, J=4.07 Hz, H-2′a), 1.18 (d, 3H, J=6.2 Hz, H-6′), 1.12 (s, 9H, t-Bu).

(ii) WP 750 FIG. 21

4′-O-Benzyl-4′epi-doxorubicin 3′-Deamino-3′-azido-4′-O-benzyl-14-O-tertbutyldiphenylsilyl-doxorubicin

Product was obtained according to the Procedure (A), yield 90%.

nmr (CDCl3) δ: 13.90, 13.18 (2s, 1H each, 6-OH, 11-H), 8.01 (dd, 1H,J=7.49 Hz, J=0.9 Hz, H-1), 7.76 (dd, 1H, J=J=8.11 Hz, H-2), 7.72-7.69(m, 4H), 7.47-7.33 (m, 12H, H-3 and H-aromatic), 5.49 (d, 1H, J=3.26 Hz,H-1′), 5.16 (, 1H, H-7), 4.83 (ABq, 2H, 14-CH2), 4.83 (d, 1H, J=11.14Hz, CH2Ph), 4.57 (d, 1H, J=11.28 Hz, CH2Ph), 4.27 (s, 1H, 9-OH), 4.05(s, 3H, OMe), 3.75 (q, 1H J=6.4 Hz, H-5′), 3.42-3.36 (m, 2H, H-4′, H-3),3.00 (d, 1H, J=19.09 Hz, H-10), 2.80 (d, 1H, J=19.03 Hz, H-10), 2.17(ddd, 1H, J=J=13.06 Hz, J=3.86 Hz, H-2′a), 2.1-1.95 (m, 2H, H-8a,e),1.81(dd, 1H, J=13.06 Hz, J=3.81 Hz, H-2′e), 1.09 (s, 9H, t-Bu), 0.975(d, 3H, J=6.4 Hz, H-6′).

(iii) WP 744 FIG. 22

4′-O-Benzyl-doxorubicin

Product was obtained according to the procedure B., yield 40

n.m.r (DMSO) δ: 14.00, 13.22 (2s, 1H each, 6-OH, 11-OH), 8.15 (, 2H,NH2), 7.91-7.89 (m, 2H, H-1,2), 7.65-7.63 (m, 2H, H-3), 7.47-3.30 (m,5H, H-aromatic), 5.45 (s, 1H, 14-OH), 5.40 (d, 1H, J=2.48 Hz, H-1′), 4.9(, 1H, H-7), 4.85 (, 1H, 9-OH), 4.79 (d, 1H, J=11.13 Hz, Ch2Ph), 4.57(s, 2H, 14-CH2), 4.56 (d, 1H, J=11.13 Hz, Ch2Ph), 4.23 (q, 1H, J=6.51Hz, H-5′), 3.97 (s, 3H, OMe), 3.72 (, 1H, H-4′), 3.52-3.45 (m, 1H,H-3′), 2.97 (d, 1H, J=18.24 Hz, H-10), 2.85 (d, 1H, J=18.24 Hz, H-10),2.14-2.05 (m, 2H, H-8), 1.87 (ddd, 1H, J=12.63 Hz, J=12.63 Hz, J=3.5 Hz,H-2′a), 1.71 (dd, 1H, J=12.63 Hz, J=4.38 Hz, H-2′e), 1.12 (d, 3H, J=6.48Hz, H-6′).

(iv) WP 783 FIG. 20

4′-O-Benzyl-4-demethoxydaunorubicin

The mixture of aglycone (1 mmol) HgBr2 (0.7 g), HgO (2.8 g) andmolecular sieves 4 Å (1 g) in methylene chloride (50 mL) was preparedand stirred at room temperature. Trimethylsilyl bromide (3 mmol) wasadded to the solution of sugar (2 mmol) in dichlorometahne (8 mL). Theobtained mixture was stirred at room temp., progress of the reaction wasmonitored by TLC (Toluene/acetone=8:1), until all substrate disappearedfrom reaction mixture, the solvent was removed under diminishedpressure. Hexanes (10 mL) were added to the residue. Solvent was removedagain, and the second portion of hexanes was added and then removed fromthe mixture. The addition and removal of hexanes was repeated threetimes then dry residue was dissolved in dichloromethane (10 mL).Obtained solution was added in three portions to the earlier preparedsolution of aglycone and mercuric salts. The mixture was stirred at roomtemperature for 10 min. (reaction was controlled by TLC, then it wasdiluted with methylene chloride (50 mL), and filtered through Celite.Filtrate was washed with 10% water soln. of KI, then twice with water,and it was dried over Na2SO4. The crude product was purified by columnchromatography (Eluent: toluene, toluene:acetone=100:1, 98:2). Productcontained azido group in position 3′ (1 mmol) was dissolved in THF (8mL). Triphenyl phosphine (2 mmol) was added to this solution, andobtained mixture was stirred at room temperature overnight. Progress ofthe reaction was controlled by TLC. The reaction mixture was thendiluted with chloroform, then 2N NH3 in methanol (2 mL) was added tothis solution. Everything was stirred together for 1 hr, then thesolution was washed with water until neutral, then with brine, and driedover Na2SO4. Crude product was purified by column chromatography(eluent:chloroform, chloroform:methanol=98:2, 95:5). Pure free amine wasdissolved in methanol (4 mL), 1solution of HCl in methanol (1.5 mL)followed by diethyl ether were added to this solution. Obtained solidhydrochloride was filtered off and washed with diethyl ether to removeexcess of acid. When the filtrate was neutral, solid hydrochloride wasprecipitated from methanol/diethyl ether, gave analytically pureproduct. (Yield 40%).

1H n.m.r (CDCl3), δ: 8.35-8.33 (m, 2H, H-1,4), 7.85-7.80 (m, 2H, H-2,3),7.42-7.29 (m, 5H, aromatic), 5.50 (d, 1H, J=3.55 Hz, H-1′), 5.28 (, 1H,H-7), 4.82 (d, 1H, J=11.52 Hz, aromatic), 4.76 (, 2H, NH2), 4.67 (d, 1H,J=11.51 Hz, aromatic), 4.10 (q, 1H, J=6.58 Hz, H-5′), 3.44 (, 1H, H-4′),3.24 (dd, 1H, J=19.0 Hz, J=1.59 Hz, H-10), 3.05-2.95 (m, 1H, H-3′), 3.00(d, 1H, J=19.0 Hz, H-10), 2.34 (d, 1H, J=14.77 Hz, H-8), 2.07 (dd, 1H,J=14.74 Hz, J=4.0 Hz, H-8), 1.85 (ddd, 1H, J=J=13.01 Hz, J=3.1 Hz,H-2′a), 1.70 (dd, 1H, J=13.09 Hz, J=4.53 Hz), 1.32 (d, 3H, J=6.57 Hz,H-6′).

(v) WP 799 FIG. 17

4′-(2,6-difluoro-benzyloxy)-doxorubicine

Product was obtained according to the procedure A and B, yield 45% 1Hn.m.r (CDCl3), δ: 7.9 (d, 1H, J=7.45 Hz, H-1), 7.72 (dd, 1H, J=7.9 Hz,J=7.9 Hz, H-2), 7.23 (d, 1H, J=7.8 Hz, H-3), 7.28-7.24 (m, 2H,aromatic), 6.89-6.86 (m, 2H, 2,6 aromatic), 5.44 (d, 1H, J=2.5 Hz,H-1′), 5.26 (, 1H, H-7), 4.76 (ABq, 2H, aromatic), 4.71 (s, 4H, aromaticand 9-OH), 4.10-3.98 (m, 1H, H-5′), 4.02 (s, 3H, OMe), 3.42 (, 1H,H-4′), 3.20 (d, 1H, J=19 Hz, H-10), 2.97 (d, 1H, J=19 Hz, H-10),3.01-2.90 (m, 1H, H-3′), 2.27 (d, 1H, J=14.8 Hz, H-8), 2.07 (dd, 1H,J=14 Hz, J=3.7 Hz, H-8), 1.75-1.64 (m, 2H, H-2′a, H-2′e), 1.29 (d, 3H,J=6.5 Hz, H-6′).

(vi) WP 797 FIG. 18

4′-(4-Fluoro-benzyloxy)-4-demethoxydaunorubicine

Product was obtained according to the procedure A and B, yield 60% 1Hn.m.r (CDCl3), δ: 8.3-8.27 (m, 2H H-1, H-4), 7.81-7.76 (m, 2H, H-2.3),7.36-7.29 (m, 2H, 4F-Ph-CH2), 7.02-6.96 (m, 2H, 4F-Ph-CH2), 5.46 (d, 1H,J=3.5 Hz, H-1′), 5.24 (, 1H, H-7), 4.66 (ABq, 2H, 4F-Ph-CH2), 4.07 (q,1H, J=6.59 Hz, H-5′), 3.39 (, 1H, H-4′), 3.20 (dd, 1H, J=19 Hz, J=1.9Hz, H-10), 2.95 (d, 1H, J=19 Hz, H-10), 3.0-2.9 (m, 1H, H-3′), 2.37 (s,3H, OMe), 2.30 (d, 1H, J=14 Hz, H-8), 2.03 (dd, 1H, J=14 Hz, J=4.2Hz,H-8), 1.88 (ddd, 1H, J=13 Hz, J=13 Hz, J=3.93 Hz, H-2′a), 1.67 (dd, 1H,J=13 Hz, J=4.6 Hz, H-2′e), 1.26 (d, 3H, J=6.6 Hz, H-6′).

B. Synthesis of 3-azido-2,3,6-trideoxyhexopyranoses (3-azido-daunosamineand 3 azidoacosamine), Sugars Used in the Synthesis of 4′-O-benzylatedAnalogs of 3′ amino-anthracyclines.

(vii) WP 219 FIG. 40

4-O-acetyl-3-azido-1-O-t-butyldimethylsilyl-2,3,6-trideoxy-b-L-arabinohexapyranose

The mixture of 3,4-di-O-acetyl-L-rhamnal (1 mol) and water (2700 mL) washeated to 70° C., and it was stirred at this temperature until substrateis hydrolyzed (TLC). After substrate disappearance the mixture wascooled down to 0° C., then sodium azide (2.2 mol) followed by aceticacid (85 mL) were added to the mixture, and it was stirred at 0° C. for2 hr. Second portion of sodium azide (2.2 mol) and acetic acid (85 mL),and methylene chloride (1500 mL) were added to the reaction mixture, andit was stirred at room temperature overnight. The organic layer wasseparated, the water layer was extracted with dichloromethane (3×500mL). Combined organic solutions were washed with water until neutral,then dried over Na2SO4. The drying agent and solvent were removed, andobtained product, light yellow oil was dried, gave crystals (0.9 mol).Obtained mixture of azides was dissolved in N,N dimethylformamide (300ml), then imidazole (256 g), and tertbutyldimetylsilyl chloride (185 g)were added. The mixture was stirred at room temperature overnight, thenit was diluted with water (600 mL) and extracted with hexanes (4×500mL). The combined organic extracts were washed with water until neutral,then with brine, and it was dried over Na2SO4. The drying agent andsolvent were removed and crude product—light yellow oil was dried withvacuum pump overnight. Product after silylation was dissolved inmethanol (1400 mL), and cooled down to 0° C. then sodium methanolate (1Msolution in methanol) (14 ml) was added. The reaction mixture wasstirred at 0° C. for 3 hr., then 1 N water solution of HCl (14 ml) wasadded. The mixture was diluted with water (1400 mL), and extracted withhexanes (3×500 mL), then with dichloromethane (3×500 mL). The hexanesextracts were combined and washed with water until neutral, then withbrine, and it was dried over Na2SO4. Crude product was purified bycolumn chromatography, using as an eluent: hexanes, hexanes:ethylacetate=98:2, gave colorless oil4-O-acetyl-3-azido-1-O-t-butyldimethylsilyl-2,3,6-trideoxy-β-L-arabinohexopyranose (158 g; 0.48 mol). [a]D 1.46° (c=1.045, chloroform).

n.m.r (CDCl3), d: 4.82 (dd, 1H, J=9.3 Hz, J=1.9 Hz, H-1), 4.67 (t, 1H,J=J=9.8 Hz, H-4), 3.50 (ddd, 1H, J=4.9 Hz, J=12.8 Hz, J=9.8 Hz, H-3),3.44 (qd, 1H, J=6.3 Hz, J=9.3 Hz, H-5), 2.20 (ddd, 1H, J=12.8 Hz, J=4.9Hz, J=1.9 Hz, H-2e), 2.16 (s, 3H, OAc), 1.69 (ddd, 1H, J=12.8 Hz, J=12.8Hz, J=9.3 Hz, H-2a), 1.21 (d, 3H, J=6.3 Hz, H-6), 0.90 (s, 9H, t-BuSi),0.13, 0.11 (s, 3H, Me2Si). Anal elem calc for C14H27N3O4Si: C51.04, H8.26, N 12.75. Found: C 51.3, H 8.33, N 12.47.

(viii) WP 460 FIG. 40

3-azido-1-O-t-butyldimethylsilyl-2,3,6-trideoxy-b-L-arabino-hexapyranose

Obtained in previous step sugar (100 g) was dissolved in methanol (500mL), then solid potassium carbonate (100 g) was added to this solution.The mixture was stirred at room temperature. After 0.5 hr reaction wascompleted, solid salts were filtered off. Organic solution was dilutedwith water (300 mL), and extracted with hexanes (3×300 mL), Combinedextracts were washed with water until neutral, dried over sodiumsulfate. The drying agent and solvents were removed gave pure product(white crystals), 82.8 g, yield 95%; [a]D+28.9° (c=1.3, chloroform). 1Hn.m.r (CDCl3), d: 4.83 (dd, 1H, J=9.3 Hz, J=2.0 Hz, H-1), 3.42 (q, 1H,J=4.8 Hz, J=12.6 Hz, J=9.6 Hz, H-3), 3.35 (dq, 1H, J=6.1 Hz, J=9.0 Hz,H-5), 3.17(td, 1H, J=9.6 Hz, J=9.0 Hz, J=3.6 Hz, H-4), 2.30 (d, 1H,J=3.6 Hz, 4-OH), 2.21 (ddd, 1H, J=12.6 Hz, J=4.8 Hz, J=2.0 Hz, H-2e),1.68 (ddd, 1H, J=12.6 Hz, J=12.6 Hz, J=9.3 Hz, H-2a), 1.34 (d, 3H, J=6.1Hz, H-6), 0.92 (s, 9H, t-Bu), 0.14, 0.13 (2s, 1H each, Me2Si). Anal elemcalc for C12H25N3O3Si: C 50.14, H 8.77, N 14.62. Found: C 50.20, H 8.78,N 14.54.

C. Alternative method for Resolution of Mixture 3-azido-L-arabino- and3-azido L-ribo-hexapyranoses.

3-Azido-2,3,6-trideoxy-L-arabino- and 3-azido-2,3,6-trideoxy-L-ribohexapyranses obtained by hydrolysis of 3,4-do-O-acetyl-L-rhamnal andsubsequent Michael addition of HN3, were separated by crystallization.

(ix) WP 417 FIG. 40

Synthesis of 3-azido-4-O-benzyl-2,3,6-trideoxy-L-lyxohexapyranoside

t-Butyldimethylsilyl 3-azido-2,3,6-trideoxy-L-arabinohexopyranose(WP460) (18.0 g, 62.7 mmol) was added to a solution of dry pyridine (38ml) dichloromethane (1200 ml). The solution was purged with argon andcooled to −40° C. To this vigorous stirred mixture, solution oftrifluoromethanesulfonic anhydride (38 ml, 63.4 g. 225 mmol) inmethylene chloride (100 ml) was added dropwise through septum, during1.5 h. The reaction mixture was allowed to warm up to the roomtemperature, then it was diluted with methylene chloride (300 ml) andwashed with sodium acetate (10% aqueous solution) and phosphate buffer(pH 8) (2×200 ml). The solvent was evaporated twice with toluene (200ml). The oily residue was dissolved in dry dimethylformamide (25 ml) andtetrabutylmmonium acetate (22.0 g, 71 mmol) was added to this solution.The mixture was stirred for 1 h at room temperature. After the completedisappearance of the triflate (TLC) the mixture was diluted with water(300 ml), and extracted with ethyl acetate (3×250 ml). Combined extractswere washed with 5% NaHCO3, and water until neutral. The organic layerwas then dried over anhydrous sodium sulfate. Crude product was purifiedby column chromatography gave pure WP417 (15.5 g, 47.1 mmol). Yield:75.1%; [a]D +5.3° (c=1.3, methylene chloride); 1H n.m.r (CDCl3), d: 5.06(d, 1H, J=3.1 Hz, H4), 4.78 (dd, 1H, J=8.4 Hz, J=2.8 Hz, H-1), 3.60 (q,1H, J=6.44 Hz, H-5), 3.42 (ddd, 1H, J=12.1 Hz, J=5.3 Hz, J=3.2 Hz, H-3),2.18 (s, 3H, OAc), 2.00-1.86 (m, 2H, H-2a, H-2e), 1.18 (d, 3H, J=6.5 Hz,H-6), 0.91 (s, 9H, t-Bu), 0.14, 0.12 (2s, 3H each, Me2Si). Anal Calcfor: C14H27N3O4Si: H, 8.26, C, 51.04, N, 12.75. Found: H, 8.31, C,51.22, N, 12.68

(x) WP 418 FIG. 40

t-Butyldimethylsilyl 3-azido-2,3,6-trideoxy-β-L-lyxo hexapyranose

Obtained in previous step sugar WP471 (15.5 g) was dissolved in methanol(100 ml), then solid potassium carbonate (15.5 g) was added to thissolution. The mixture was stirred at room temperature. Progress of thereaction was monitored by T.L.C. After 0.5 hr reaction was completed,solid salts were filtered off. Organic solution was diluted with water(100 ml), and extracted with hexanes (3×200 ml), Combined extracts werewashed with water until neutral and dried over sodium sulfate. Thedrying agent and solvents were removed gave pure product (whitecrystals), 13.25 g Yield: 98%

mp 74-76° C., [a]D −4.1° (c=1.8, dichloromethane), 1H n.m.r (CDCl3), d:4.74 (dd, 1H, J=9.1 Hz, J=2.4 Hz, H-1), 3.60 (dd, 1H, J=2.7 Hz, J=8.4Hz, H-4), 3.50 (q, 1H, J=6.5 Hz, H-5), 3.30 (ddd, 1H, J=2.9 Hz, J=12.7Hz, J=2.7 Hz, H-3), 2.03 (d, 1H, J=8.4 Hz, 4-OH), 1.99 (ddd, 1H, J=12.7Hz, J=2.9 Hz, J=2.4 Hz, H-2e), 1.84 (td, 1H, J=12.7 Hz, J=12.7 Hz, J=9.1Hz, H-2a), 1.29 (d, 3H, J=6.5 Hz, H-6), 0.90 (s, 9H, t-Bu), 0.13, 0.11(s, 3H each, Me2Si). Anal Calc for: C12H25N3O3Si: H, 8.77, C, 50.14, N,14.62. Found: H, 8.80, C, 50.11, N, 14.52

(xi) WP 819 FIG. 26

1-O-t-butyldimethylsilyl-3-Azido-4-O-benzylo-2,3,6-trideoxy-β-L-lyxohexapyranose

Sodium hydride (1 g of 60% suspension in mineral oil) was added to acooled to 0° C. solution of WP 418 (3.9 g, 13.5 mmol) in dry N,Ndimethylformamide (40 ml). The reaction mixture was stirred for 15 min,then solution of benzyl bromide (2.97 ml, 4.27 g, 25 mmol) was added.The stirring continued for 0.5 h at 0° C., then mixture was allowed towarm up to room temperature. The reaction mixture was poured into theice/water and extracted with ethyl acetate (3×100 ml). The combinedextracts were washed with water, then with saturated solution of sodiumbicarbonate and dried over magnesium sulfate. The pure product (3.145 g,8.34 mmol) was separated by column chromatography. Yield: 61.8%. 1Hn.m.r (CDCl3) d: 7.4-7.2 (m, 5H, H-aromatic), 4.84 (d, 1H, J=11.4.Hz,H-aromatic), 4.70 (dd, 1H, J=9 Hz, J=2.19 Hz, H-1), 4.61 (d, 1H, J=11.4Hz, Ch2Ph), 3.39 (q, 1H, J=6.1 Hz, H-5), 3.33 (d, 1H, J=2.67 Hz, H-4),3.25 (ddd, 1H, J=4.46 Hz, J=12.8 Hz, J=2.67 Hz, H-3), 2.03 (ddd, 1H,J=12.3 Hz, J=12.8 Hz, J=9 Hz, H-2a), 1.90 (ddd, 1H, J=12.3 Hz, J=4.46Hz, J=2.19 Hz, H-2e), 1.15 (d, 3H, J=6.1 Hz, H-6), 0.90 (s, 9H, t-Bu),0.13, 0.11 (s, 3H each, Me2Si).

D. Direct Synthesis of 3-azido-daunosamine.

(xii) WP 417 FIG. 40

4-O-acetyl-3-Azido-1-O-t-butyldimethylsilyl-2,3,6-trideoxy-β-L-lyxohexapyranose

The mixture of 3,4-di-O-acetyl-fucal (1 mol) and water (2700 mL) washeated to 80° C., and it was stirred at this temperature until all3,4-di-O-acetyl-fucal hydrolysed (TLC). After all substrate disappearedfrom reaction mixture, the mixture was cooled down to 0° C., then sodiumazide (2.2 mol) followed by acetic acid (85 mL) were added to themixture, and it was stirred at 0° C. for 2 hr. Second portion of sodiumazide (2.2 mol) and acetic acid (85 mL), and dichloromethane (1500 mL)were added to the reaction mixture, and it was stirred at roomtemperature overnight. The organic layer was separated, the water layerwas extracted with dichloromethane (3×500 mL). Combined organicsolutions were washed with water until neutral, then dried over Na2SO4.The drying agent and solvent were removed, and obtained product mixture,a light yellow oil was dried, to gave crystals (0.89 mol). Obtainedmixture of azides was dissolved in dimethylformamide (300 mL), thenimidazole (256 g), and tert butyldimetylsilyl chloride (185 g) wereadded. The mixture was stirred at room temperature overnight, then itwas diluted with water (600 mL) and extracted with hexanes (4×500 mL).The combined organic extracts were washed with water until neutral, thenwith brine, and it was dried over Na2SO4. The drying agent and solventwere removed and crude product, light yellow oil was dried overnight.Product after silylation was dissolved in methanol (1400 mL), and cooleddown to 0° C. then sodium methanolate (1 M solution in methanol) (14 mL)was added. The reaction mixture was stirred at 0° C. for 3 hr., then 1 Nwater solution of HCl (14 mL) was added. The mixture was diluted withwater (1400 mL), and extracted with hexanes (3×500 mL), then withmethylene chloride (3×500 mL). The hexanes extracts were combined andwashed with water until neutral, then with brine, and it was dried overNa2SO4. Crude product was purified by column chromatography, usinghexanes, hexanes:ethyl acetate=98:2 as eluent, gave analytically pureproduct (48.5 g; 0.168 mol); [a]D +5.3° (c=1.3, chloroform), 1H n.m.r(CDCl3), d: 5.06 (d, 1H, J=3.1 Hz, H-4), 4.78 (dd, 1H, J=8.4 Hz, J=2.8Hz, H-1), 3.60 (q, 1H, J=6.44 Hz, H-5), 3.42 (ddd, 1H, J=12.1 Hz, J=5.3Hz, J=3.2 Hz, H-3), 2.18 (s, 3H, OAc), 2.00-1.86 (m, 2H, H 2a, H-2e),1.18 (d, 3H, J=6.5 Hz, H-6), 0.91 (s, 9H, t-Bu), 0.14, 0.12 (2s, 3Heach, Me2Si). Anal elem calc for: C14H27N3O4Si: C, 51.04, H, 8.26, N,12.75. Found: C, 51.22, H, 8.31, N, 12.68.

E. 4′-O-benzylated-3′hydroxy-doxorubicin analogs:

(xiii) WP 568 FIG. 35

Synthesis of3-Oacetyl-4-Obenzyl-3-deamino-14-Ot-butyldiphenylsilyl-doxorubicin

A mixture of 14-O-t-butyldiphenylsilyl-adriamycinone (0.84 g, 1.29mmol), 3-O-acetyl-4-O-benzyl-L-fucal (0.84 g, 3.2 mmol),triphenylphosphine hydrobromide (0.055 g, 0.16 mmol) in dichloromethane(18 mL) was stirred for 48 hr. During this time additional portion of3-O-acetyl-4-O-benzyl-L-fucal (0.175 g, 0.7 mmol) was added and stirredfor 24 hours. The reaction mixture was diluted with dichloromethane (200mL), washed with water and dried with anhydrous Na2SO4. The product waspurified by chromatography on silicagel column usingdichloromethane:acetone (95:5 vol.) to give3-O-acetyl-4-O-benzyl-3-deamino-14-O-t-butyldiphenylsilyl-doxorubicin(WP 568), (1.0 g, 1.09 mmol). Yield: 84%. [a] D 116.84o (c=0.0354,chloroform:methanol 1:1). 1H n.m.r (CDCl3), d: 13.94 (s, 1H, OH-6),13.24 (s, 1H, OH-11), 8.1 (d, 1H, J=7.8 Hz, H-1), 7.68-7.85 (m, 5H,J=8.1 Hz, from silyl, H-2), 7.5-7.2 (m, 12H, J=7.3 Hz, from silyl,Ch2Ph, H-3), 5.49 (d, 1H, J=3.9 Hz, H-1), 5.19 (bs, 1H, H-7), 5.0-4.78(m, 3H, J=19.8 Hz, H-14, H-3), 4.7 (d, 1H, J=11.6 Hz, Ch2Ph), 4.59 (d,1H, J=11.8 Hz, Ch2Ph), 4.29 (s, 1H, OH-9), 4.07 (s, 3H, CH3O), 3.90 (q,1H, J=6.5 Hz, H-5 ), 3.60 (bs, 1H, H4 ), 3.02 (d, 1H, J=19.3 Hz,H-10eq), 2.85 (d, 1H, J=19.1 Hz, H-10ax), 2.3-1.95 (m, 3H, H-8, H-2a),1.93 (s, 3H, CH3CO), 1.8 (dd, 1H, J=3.8 Hz, J=12.7 Hz, H-2e ), 1.43 (s,9H, tBu), 1.07 (d, 3H, J=6.5 Hz, H-6).

(xiv) WP 569 FIG. 35

Synthesis of4-Obenzyl-3-deamino-3-hydroxy-14-Ot-butyldiphenylsilyl-doxorubicin

3-O-Acetyl-4-O-benzyl-3-deamino-14-O-t-butyldiphenylsilyl-doxorubicin(WP 568) (1.0 g, 1.09 mmol) was dissolved in mixture of tetrahydrofuran(20 mL) and methanol (80 mL), and then anhydrous potassium carbonate(1.0 g, 7.2 mmol) was added. The reaction mixture was stirred vigorouslyat room temperature for 2 h, until substrate disappeared. Then reactionmixture was diluted with dichloromethane (300 mL) and poured into 0.05 NHCl (100 mL), extracted with dichloromethane, washed with water, driedwith anhydrous Na2SO4. The solvent was evaporated and the residue waschromatographed using dichloromethane:acetone (9:1 vol.) to give4-O-benzyl-3-deamino-3-hydroxy-14-O-t-butyldiphenylsilyl-doxorubicin (WP569) (0.6 g, 0.687 mmol). Yield: 63%. [a] D 52.94o (c=0.022,chloroform:methanol 1:1). 1H n.m.r (CDCl3), d: 13.93 (s, 1H, OH-6),13.23 (bs, 1H, OH-11), 8.02 (d, 1H, J=7.6 Hz, H-1), 7.82-7.67 (m, 5H,from silyl, J=8.2 Hz, H-2), 7.45-7.25 (m, 12H, J=8.3 Hz, from silyl,Ch2Ph, H-3), 5.47 (d, 1H, J=3 Hz, H-1), 5.19 (bs, 1H, H-7), 4.95 (d, 1H,J=19.8 Hz, H-14), 4.84 (d, 1H, J=19.8 Hz, H-14), 4.80 (d, 1H, J=11.6 Hz,Ch2Ph), 4.64 (d, 1H, J=11.6 Hz, Ch2Ph), 4.43 (s, 1H, OH-9), 4.07 (s, 3H,CH3O), 3.92 (q, 1H, J=6.5 Hz, H-5), 3.70(bs, 1H, H-3), 3.45 (d, 1H,J=2.3 Hz, H-4), 3.0 (d, 1H, J=19 Hz, H-10eq), 2.83 (d, 1H, J=19 Hz,H-10ax), 2.35 (s, 1H, OH), 2.12-195 (m, 2H, H-8), 1.92-1.7 (m, J=3.8 Hz,H-2), 1.13 (s, 12H, tBu), 1.11 (s, 3H, H-6).

(xv) Synthesis of 4-Obenzyl-3-Deamino-3-Hydroxy-Doxorubicin—WP 727 FIG.23

4-O-Benzyl-3-deamino-3-hydroxy-14-O-t-butyldiphenylsilyl-doxorubicin (WP569) (0.6 g, 0.687 mmol) was dissolved in THF (33 mL), and a reagent (25mL)/made from THF (24 mL), dichloromethane (12 mL), pyridine (0.66 mL),1.0 M Bu4NF (0.6 mL)/was added, and stirred vigorously until substratedisappear. Then the reaction mixture was poured into solution containingice (50 g), 0.1 N HCl (100 mL), dichloromethane (100 mL). Organic layerwas separated and water layer was extracted with dichloromethane (3×50mL). The combined organic layers were washed with water, dried withanhydrous Na2SO4, evaporated, and chromatographed on silicagel columnusing dichloromethane:acetone (7:3 vol.) to give pure product4-O-benzyl-3-deamino-3-hydroxy-doxorubicin (WP 570) (0.34 g, 0.536mmol). Yield: 78%. [a] D 101.00o (c=0.029, chloroform:methanol 1:1). 1Hn.m.r (CDCl3) d: 13.93 (s, 1H, OH-6), 13.22 (s, 1H, OH-11), 8.02 (d, 1H,J=8 Hz, H-1), 7.77 (t, 1H, J=8 Hz, H-2), 7.38 (m, 6H, H-aromatic, H-3),5.55 (d, 1H, J=3.2 Hz, H-1), 5.26 (s, 1H, H-7), 4.83 (dd, 1H, J=11.8 Hz,H-14), 4.7-4.65 (m, 4H, J=2.75 Hz, H-14, H-aromatic, OH-9), 4.07 (s, 3H,CH3O), 4.03 (q, 1H, J=6.6 Hz, H-5 ), 3.9-3.78 (m, 1H, H-3), 3.57 (d, 1H,J=2.52 Hz, H4), 3.25 (dd, 2H, J=1.5 Hz, J=18.7 Hz, H-10eq), 3.01 (d, bs,3H, J=18.8 Hz, H-10ax, OH-3), 2.32 (bd, 1H, J8e-8a=14.8 Hz, H-8eq), 2.13(dd, 1H, J=3.9 Hz, J8a-8e=14.7 Hz, H-8ax), 1.95 (dt, 1H, J=4 Hz, J=12.8Hz, H-2a), 1.84 (dd, 1H, J=4.8 Hz, J=12.8 Hz, H-2e), 1.33 (d, 3H, J=6.5Hz, H-6).

(xvi) WP 571 FIG. 38

Synthesis of4-Obenzyl-3-deamino-3-Otriethylsilyl-14-Opentanoyl-doxorubicin

A mixture of 14-O-pentanoyl-adriamycinone (0.6 g, 1.2 mmol),4-O-benzyl-3-O-triethylsilyl-L-fucal (1.1 g, 3.3 mmol), sieves 4 (0.5g), TPHB (0.06 g, 0.18 mmol) in dichloromethane (20 mL) was stirredovernight at room temperature. Then the reaction mixture was dilutedwith dichloromethane (100 mL) and washed with water, dried withanhydrous Na2SO4; the solvent was evaporated, and the residue waschromatographed on silicagel columusing dichloromethane:acetone (98:2vol.) to give4-O-benzyl-3-deamino-3-O-triethylsilyl-14-O-pentanoyl-doxorubicin (0.89g, 1.07 mmol). Yield: 89%. 1H n.m.r (CDCl3), d: 13.94 (s, 1H, OH-6),13.25 (s, 1H, OH-11), 8.03 (d, 1H, J=7.8 Hz, H-1), 7.77 (t, 1H, J=8 Hz,H-2), 7.43-7.26 (m, 6H, J=7.2 Hz, H-aromatic, H-3), 5.55 (d, 1H, J=3.7Hz, H-1), 5.31 (bs, 1H, H-7), 5.32 (d, 1H, J=18.2Hz, H-14), 5.12 (d, 1H,J=18.2 Hz, H-14), 4.98 (d, 1H, J=12.6 Hz, Ch2Ph), 4.92 (s, 1H, OH-9),4.67 (d, 1H, J=12.7 Hz, Ch2Ph), 4.08 (s, 3H, CH3O), 4.02-3.90 (q, m, 2H,J5=6.5 Hz, H-5, H-3 ), 3.45 (bs, 1H, H-4), 3.28 (d, 1H, J=19 Hz,H-10eq), 3.03 (d, 1H, J=19 Hz, H-10ax), 2.50-2.38 (m, 4H, J=7.4 Hz, CH2from pentanoyl, H-8), 2.22 (dt, 1H, J=3.1 Hz, J=12.7 Hz, H-2a ), 2.06(dd, 1H, J=3.8 Hz, J=12.7 Hz, H-2e), 1.77-1.62 (m, 2H, J=7.4 Hz, CH2from pentanoyl), 1.48-1.35 (m, 2H, J=7.5 Hz, CH2 from pentanoyl), 1.23(d, 3H, J=6.5 Hz, H-6 ), 0.95 (t,t, 12H, J=7.8 Hz, CH3 from pentanoyl,(Et)3Si), 0.58 (q, 6H, J=8 Hz, (Et)3Si).

(xvii) WP 571 FIG. 38

Synthesis of 4-Obenzyl-3-Deamino-3-Hydroxy-14-Opentanoyl-Doxorubicin

4-O-Benzyl-3-deamino-3-O-triethylsilyl-14-O-pentanoyl-doxorubicin (0.715g, 0.86 mmol) was dissolved in THF (15 mL), and then 0.1 N HCl (10 mL)was added. Reaction mixture was stirred at room temperature for 1 huntil substrate disappeared. Then the reaction mixture was diluted withwater (30 mL); a precipitate was filtered, washed with water and driedat 100 C under diminished pressure overnight to give pure3-deamino-4-O-benzyl-3-O-hydroxy-14-O-pentanoyl-doxorubicin (WP 571),(0.56 g, 0.78 mmol). Yield: 91%. 1H n.m.r (CDCl3), d: 13.91 (s, 1H,OH-6), 13.18 (s, 1H, OH-11), 7.99 (d, 1H, J=7.7 Hz, H-1), 7.75 (t, 1H,J=8 Hz, H-2), 7.46-7.29 (m, 6H, H-aromatic, H-3), 5.54 (d, 1H, J=3.2 Hz,H-1), 5.32 (d, 1H, J=18.1 Hz, H-14), 5.25 (bs, 1H, H-7), 5.10 (d, 1H,J=18.2 Hz, H-14), 4.85 (d, 1H, J=11.7 Hz, Ch2Ph), 4.74 (s, 1H, OH-9),4.69 (d, 1H, J=11.7 Hz, Ch2Ph), 4.12-4.00 (q, s, 4H, H-5, CH3O),3.90-3.70 (m, 1H, H-3), 3.58 (d, 1H, J=2 Hz, H-4), 3.25 (d, 1H, J=18.7Hz, H-10eq), 2.97 (d, 1H, J=18.7 Hz, H-10ax), 2.52-2.38 (m, 3H, J=7.4Hz, CH2 from pentanoyl, H-8), 2.08 (dd, 1H, J=3.8 Hz, J=14.9 Hz, H-8),1.95 (dt, J=4 Hz, J=12.9 Hz, H-2a), 1.89-1.82 (dd, 1H, J=5 Hz, J=12.9Hz, H-2e), 1.75 (d, 1H, J=9.4 Hz, OH-3), 1.75-1.65 (m, 2H, J=7.4 Hz, CH2from pentanoyl), 1.48-1.3 (m, 2H, J=7.4 Hz, CH2 from pentanoyl), 1.36(d, 3H, J=6.5 Hz, H-6), 0.95 (t, 3H, J=7.4 Hz, CH3 from pentanoyl).

EXAMPLE 3 Novel Method for the Synthesis of 4-O-Benzylated Sugars

Described herein is a method for the preparation of the 4-O-benzylatedsugars, used as precursors in the synthesis of4-O-benzylated-3-hydroxy-anthracyclines like WP727, WP571, WP794, orWP793. This is a novel method superior to other methods describedearlier. A direct selective alkylation of acetylated glycals is usedwhich gives almost exclusively products alkylated at C-4. The inventorsapproach is different from other known methods in the following ways:(1) a one step approach wherein a fully blocked compound is alkylatedallowing for an easier and simpler synthesis versus the standard methodof alkylating free hydroxyl groups, (2) because the process is selectivethe major products are 4-O-alkylated compounds. In this method, thefirst step of the reaction is a selective deblocking (deacylation) ofthe hydroxyl at C-4 followed by a rapid alkylation of this free hydroxylgroup. In contrast, alkylation of unblocked (hydroxy compounds) glycalsgives mixtures of mono-benzylated and dibenzylated products and4-O-benzylated-glycals are obtained in lower yields.

Such 4-O-benzylated glycals can be used directly towards synthesis ofthe 4-O-benzylated anthracycline analogs described in the previousexample, using electrophilic addition to glycals in the presence oftriphenylphosphine hydrobromide (TPHB), for example the synthesis ofWP727.

The inventors method works well not only for the 6-deoxy-glycals likefucal and rhamnal but to also for glycals like D-glucal or D-galactalhaving hydroxyl groups at C-3, 4, and 6 positions. Direct alkylation of3,4,6-tri-O-acetylated-D-glucal or -D-galactal give4-O-benzylated-glycals as a the main product. Therefore, this method isof general use.

More importantly, the inventors found that these reactions can becontinued in the same vessel to the next step to synthesize3-hydroxy-4-O-benzylated glycals. For this the other acetyl groups areremoved by the addition of methanol to the reaction mixture. Methanolforms in situ sodium methoxide, which in turn deacetylates all hydroxylgroups. Described below are the experimental procedures and data for the4-O-benzylated substrates which are either acetylated or bear freehydroxyl groups, and data for other 4-O-benzylated sugars to prove thegeneral nature of the inventors approach.

This method can be used to synthesize 4-O-benzylated products and alsoother 4-O-alkylated products. The 4-O-benzylated glycals can then beused directly in the coupling reactions with proper aglycon likedaunomycinone or can be first transformed into 4-O-alkylated1-O-silylated hexopyranoses which can further be coupled using a varietyof other coupling methods as for example in the synthesis of WP793 andWP794.

A. Synthesis of 4-O-Alkylated-Glycals: FIG. 39

1. Examples of Selective Benzylation of Glycals at the C-4 Position

(i) Selected procedure for 4-O-benzyl-L-rhamnal exemplifying generalprocedure. To a solution of NaH (2.5 g, 0.063 mmol) in DMF (30 mL)cooled to ?C 3,4-di-O-acetyl-L-rhamnal (6.5 g, 0.03 mmol) was added. Thereaction mixture was stirred for 15 min, and benzyl chloride (6 mL,0.046 mmol) was added. Then the reaction was allowed to warm up to roomtemperature and stirred until TLC showed complete consumption ofstarting material. The reaction was cooled to 0° C. methanol (10 ml) wasadded, with continous stirring for 10 min. The reaction mixture was thenpoured into a solution containing 1 N HCl (10 mL), ice (20 g), and ethylacetate (50 mL). The organic phase was separated and aqueous phase wasextracted with ethyl acetate (3×50 mL). The combined organic solutionwas washed with water, dried with anhydrous Na2SO4, and concentrated togive an oily-solid product that was crystallized fromdichloromethane/hexane.

(ii) 4-OBenzyl-L-rhamnal Yield: 73%, mp 110-110.5 C. [a] D −44.12o(c=1.05, ethyl acetate). 1H n.m.r (CDCl3), d: 7.6-7.0 (m, 5H,H-aromatic), 6.32 (dd, 1H, J=5.7 Hz, H-1), 4.90-4.72 (dd, 2H, J=11.8 Hz,Ch2Ph), 4.69 (dd, 1H, J1.2=5.7 Hz, J2.3=2 Hz, H-2), 4.34 (m, 1H, H-3),3.97-3.85 (dq, 1H, J5.4=9.4 Hz, J5.6=6.3 Hz, H-5), 3.32-3.22 (dd, 1H,J4.5=9.4 Hz, H-4), 1.70 (d, 1H, J=5.8 Hz, OH), 1.40 (d, 3H, J6.5=6.3 Hz,H-6).

(iii) 4-OBenzyl-L-fucal Yield: 63%, mp 77.0-77.5 C. [a] D 13.3o (c=1.18,chloroform). 1H n.m.r (CDCl3), d: 7.55-7.30 (m, 5H, H-aromatic), 6.36(dd, 1H, J1.2=6 Hz, J1.3=0.9 Hz, H-1), 4.8 (m, 2H, H-aromatic),5.75-5.68 (m, 1H, H-2), 4.45-4.35 (m, 1H, H-3), 4.08 (q, 1H, J5.6=6.6Hz, H-5), 3.68 (d, 1H, J=5.1 Hz, H-4), 2.23 (d, 1H, J=10 Hz, OH), 1.35(d, 1H, J6.5=6.6 Hz, H-6).

(iv) 4-OBenzyl-D-glucal Yield: 72%, mp 98.5-100 C. [a] D 10.37o (c=1.4,chloroform). 1H n.m.r (CDCl3), d: 7.5-7.25 (m, 5H, H-aromatic), 6.37(dd, 1H, J1.2=5.9 Hz, J1.3=1.1 Hz, H-1), 4.95-4.78 (dd, 2H, H-aromatic),4.75 (dd, 1h, J2,1=5.9 Hz, J2,3=2.3 Hz, H-2), 4.41 (m, 1H, H-3),4.02-3.82 (m, 3H, H-6, H-5), 3.65 (t, J=6.9 Hz, H-4), 1.90 (d, J=12.7Hz, 1H, OH).

(v) 4-O-Benzyl-D-galactal Yield: 55%, mp 98.5-99° C.

2. Selected Procedure for Preparation of 3-Oacetyl-4-Obenzyl-L-rhamnalExmeplifying General Procedure.

To a solution of NaH (2.5 g, 0.063 mmol) in DMF (30 mL) cooled to 0 C3,4-di-O-acetyl-L-rhamnal was added (6.5 g, 0.03 mmol). The reaction wasstirred for 15 min, and BnCl (6 ml, 0.046 mmol) was added. Then thereaction was allowed to warm to room temperature and stirred vigorouslyuntil TLC showed complete consumption of starting material. After 1.5 hreaction mixture was poured into solution containing 1 N HCl (10 mL),ice (20 g), and ethyl acetate (50 mL). The organic phase was separatedand aqueous phase was extracted with ethyl acetate (3×50 ml). Thecombined organic solution was washed with water, dried with anhydrousNa2SO4, and concentrated to give an oil that was purified by columnchromatography on silicagel column using hexane:ethyl acetate 95:5 aseluents to give pure 3-O-acetyl-4-O-benzyl-L-rhamnal.

(vi) 3-OAcetyl-4-Obenzyl-L-rhamnal Yield: 50%, bp 200 C/0.04 mm Hg. [a]D 55.77o (c=1.15, ethyl acetate). 1H n.m.r (CDCl3) d: 7.42-7.25 (m, 5H,H-aromatic), 6.4 (d, 1H, J=6.5 Hz, H-1), 5.43-5.36 (m, 1H, H-3), 4.78(dd, dd, 3H, J2,3=2.5 Hz, J2,1=5.8 Hz, J=11.5 Hz, Ch2Ph, H-2), 4.1-4.0(m, 1H, J5,4=8.4 Hz, J5,6=6.5 Hz, H-5), 3.57-3.49 (dd, 1H, J4,5=8.2 Hz,H-4), 2.02 (s, 3H, CH3CO), 1.38 (d, 3H, J6,5=6.7 Hz, H-6).

(vii) 4-OBenzyl-3,6-di-Oacetyl-D-glucal Yield: 50%, mp 52-55.5 C. [a] D−7.34o (c=1.4, ethyl acetate). 1H n.m.r (CDCl3) d: 7.42-7.18 (m, 5H,H-aromatic), 6.42 (d, 1H, J=6.3 Hz, H-1), 5.43-5.38 (m, 1H, J=5.3 Hz,J=3.2 Hz, H-3), 4.83-4.78 (dd, 1H, J=5.9 Hz, J=3.1 Hz, H-2), 4.72 (d,1H, J=11.6 Hz, Ch2Ph), 4.64 (d, 1H, J=11.6 Hz, Ch2Ph), 4.42-4.29 (m, 2H,H-6), 4.2-4.12 (m, 1H, H-5), 3.85-3.78 (dd, 1H, J4,5=8 Hz, H-4), 2.07(s, 3H, CH3CO), 2.04 (s, 3H, CH3CO).

(viii) 4-OBenzyl-3,6-di-Oacetyl-D-galactal Yield: 59%. 1H n.m.r (CDCl3)d: 7.42-7.18 (m, 5H, H-aromatic), 6.4 (d, 1H, J=6.2 Hz, H-1), 5.53-5.4(m, 1H, J=4.4Hz, J=4 Hz, H-3), 4.85-4.77 (dd, 1H, J2,1=6.1 Hz, J2,3=4.3Hz, H-2), 4.74 (d, 1H, J=11.8 Hz, Ch2Ph), 4.55 (d, 1H, J=11.9 Hz,Ch2Ph), 4.52-4.43 (dd, 1H, J=11.7 Hz, J=8.5 Hz, H-5), 4.34-4.2 (m, 2H,J=11.7 Hz, H-6), 3.99 (t, 1H, J4,3=4 Hz, H-4), 2.09 (s, 3H, CH3CO), 2.06(s, 3H, CH3CO).

(ix) 3-OAcetyl-4-Obenzyl-L-fucal Yield: 58% bp 190 C/0.02 mm Hg. [a] D61.4 (c=0.6, chloroform).

(x) 3-OAcetyl-4-Obenzyl-L-arabinal Yield: 59%.

(xi) 3-OAcetyl-4-Obenzyl-L-xylal Yield: 39%. 1H n.m.r (CDCl3), d:7.4-7.25 (m, 5H, H-aromatic), 6.01 (d, 1H, J1,2=6.1 Hz, H-1), 5.9 (t,1H, J2,1=6.3 Hz, H-2), 5.4-5.25 (m, 1H, H-3), 4.75-4.62 (dd, 2H, J=12.2Hz, Ch2Ph, H-2), 4.15 (m, 1H, J5,5=11.6 Hz, H-5), 3.9 (m, 1H, J5,5=11.7Hz, H-5), 3.65-3.57 (m, 1H, H-4), 2.02 (s, 3H, CH3CO).

3. General procedure for preparation of3-O-silylated4-O-benzyl-L-glycals.

4-O-Benzyl-L-fucal (1.5 g, 10.4 mmol) was silylated with Et3SiCl (1.1ml, 10.4 mmol) in presence of imidazole (1.1 g, 17 mmol) in DMF (3 mL).Reaction was finished after 2 h, then water (2 mL) was added, andstirring was continued for 5 min. Then the reaction mixture wasextracted with ethyl ether (3×10 ml). The combined organic solution waswashed with water, dried with anhydrous Na2SO4, and concentrated to givean oil that was distilled on Kugelrohr 150 C/0.03 mm Hg to give pure4-O-benzyl-3-O-triethylsilyl-L-fucal (2.2 g, 6.6 mmol). Yield: 97%.

(xii) 4-O-Benzyl-3-O-t-butyldimethylsilyl-L-rhamnal. Yield: 98%.

EXAMPLE 4 Assessment of Anti-tumor Activity In Vitro

Compounds synthesized using the methods described above were testedusing a standard MTT assay (Green et al., 1984) against human carcinomasensitive (KB) and multi-drug-resistant (KBV1) cells and MCF-7 andMCF-7NVP-16 resistant cells characterized as having the MRP (multi-drugresistant associated protein) phenotype. The use of an MTT assay usingthese cells is recognized as an accepted assay for anti-tumor activityby those in the field.

Methods

In vitro Cytotoxicity against MCF-7, MCF-7/VP-16, and MCF-7/DOX celllines. In vitro drug cytotoxicities against human breast carcinomawild-type MCF-7 and MRP-resistant MCF-7/VP-16 cells were assessed byusing the MTT reduction assay, as previously reported (Green et al.,1984). The MTT dye was obtained from Sigma Chemical Co. (St. Louis,Mo.). Cells were plated in 96-well microassay culture plates (10⁴cells/well) and grown overnight at 37° C. in a 5% CO₂ incubator. Drugswere then added to the wells to achieve a final drug concentrationranging from 0.1 to 50 μg/ml. Four wells were used for eachconcentration. Control wells were prepared by adding appropriate volumesof calcium- and magnesium-free PBS (pH 7.4). Wells containing culturemedium without cells were used as blanks. The plates were incubated at37° C. in a 5% CO₂ incubator for 72 hours. Upon completion of theincubation, 20 μL of stock MTT dye solution (5 mg/mL) was added to eachwell. After a 4-hour incubation, 100 μL of buffer containing 50%N,N-dimethylformamide and 20% SDS was added to solubilize the MTTformazan. Complete solubilization was achieved by placing the plate in amechanical shaker for 30 minutes at room temperature. The opticaldensity of each well was then measured with a microplatespectrophotometer at a wavelength of 570 run. The percent cell viabilitywas calculated by the following equation:% cell viability=(OD treated wells/OD control wells)×100where OD is the mean optical density from four determinations. Thepercent cell viability values were plotted against the drugconcentrations used, and the ID₅₀ was calculated from the curve.Cytotoxicity experiments were repeated at least three times.Results and Discussion

Drug resistance, both de novo and acquired, by human tumors is currentlya major factor limiting the effectiveness of chemotherapy. Thus, for thein vitro evaluation of substituted anthracyclines having C-3′ alkylatedanthracyclines modified at benzyl ring and having4′-substituted-benzylated anthracyclines, the inventors selected twosensitive cell lines: a human carcinoma KB and MCF-7 human breastcancer, the multi-drug-resistant (MDR) counterpart of KB (KBV1carcinoma), which overexpresses MDR1 gene that encodes a membranetransport glycoprotein (P-gp), the MCF-7/VP-16 cell line thatoverexpresses the multi-drug-resistant associated protein (MRP), and theMCF-7/dox cell line which overexpresses MDR1 gene. Using this system,the inventors evaluate a drug's cytotoxic potential against human tumorsand at the same time identify compounds that might have unique activityagainst MDR tumors (Priebe et al., 1993).

Table 2 shows the in vitro evaluation of cytotoxic properties of WP791,WP790, WP786, WP785, WP784, WP778, WP775, WP774, WP758, WP757, WP756,WP755 and doxorubicin (DOX) in KB and KB-V1 cells. TABLE 2 In VitroCytotoxicity against Sensitive And MDR Tumor Cell Lines¹ KB KB-V1Compound μg/ml μg/ml RI² WP786 0.60 ± 0.20 4.9 ± 0.2 8.1 WP784 2.9 ± 0.80.8 Only one test — WP785 0.43 ± 0.05 49 ± 12 114 WP790 3.93 ± 0.81 4.6± 2.3 1.2 WP791 0.67 ± 0.25 4.6 ± 3.2 7 WP774 4.5 ± 1.3 6.33.5 1.4 WP7750.4 ± 0.2 4.1 ± 3.5 10 DOX 0.50 ± 0.0  >100 >200 WP755 1.50 ± 0.80 1.80± 0.80 1.20 WP756 2.60 ± 0.30 2.80 ± 0.30 1.08 WP757 0.67 ± 0.15 24.0036 WP758 0.67 ± 0.06 2.77 ± 0.25 4.13 WP778 0.67 ± 0.41 3.83 ± 2.75 5.7DOX 0.43 ± 0.19 109 ± 28  253¹mean of at least 4 experiments; MTT assay

Table 3 shows in vitro cytotoxic properties of WP783, WP750, WP744 anddoxorubicin (DOX) in KB, KB-V1, MCF-7 and MCF-7/VP-16 cells TABLE 3 InVitro Cytotoxicity against Sensitive and Typical MDR and MRP Tumor CellLines¹ KB KBV1 RI² MCF-7 MCF-7/VP-16 RI³ MCF-7/DOX RI⁴ EXP. 1 WP744 1.44± 0.45 2.76 ± 0.52 1.84 1.63 ± 0.5  2.3 ± 1.9 1.41 5.25 3.22 DOX 2.35 >100 >42.6 0.3 3.0 10 >100 >333 EXP. 2 WP744  1.4 ± 0.45 2.1 ± 0.51.5 1.6 ± 0.5 2.3 ± 0.9 1.4 DOX 2.1 ± 0.8 174 ± 32  82.9 0.50 ± 0.26 8.3± 0.5 16.6 WP744 0.48 ± 0.03 4.07 ± 1.85 8.5 0.37 ± 0.11 0.60 ± 0.10 1.60.89 ± 0.10 2.41 DOX 0.60 ± 0.13 >100 >167 0.53 ± 0.25 6.87 ± 0.5813 >100 >189 WP783 0.43 ± 0.21 1.1 ± 0.8 2.6 WP750 6.4 56 ± 29 8.8 DOX0.50 ± 0.0  >100 >200

Table 4 shows data for fragmented DNA expressed as percentage of totalDNA determined by quantitative apoptotic fragmentation assay in CEMleukemic cells incubated with drugs for 24 h. Results are from twoindependent studies carried out in duplicate. TABLE 4 ApoptoticFragmentation by WP744 in Comparison to Doxorubicin. Concentration WP744DOX μM % Total DNA SE % Total DNA SE 0 0 0 0.05 17.8 6.8 0.1 31.3 12.9−0.2 0.3 0.5 46.7 9.8 27.1 12.7 1 28.7 9 2 38.3 7.4

Direct comparisons of the cytotoxicity of WP791, WP790, WP786, WP785,WP784, WP783, WP778, WP775, WP774, WP758, WP757, WP756, WP755, WP750with DOX indicated that the C-3′ and C-4′ substitutions drasticallyincrease the potency of those compounds. Compound WP785 displayedsimilar profile of activity to doxorubicin and its RI was of 114 wasclearly higher than that of other analogs tested.

EXAMPLE 5 Treatment of Tumors with Analogs of Anthracyclines withSubstitutions at C-3′ or C-4′ Sugars

Treatment with the substituted anthracyclines having C-3′-alkylatedanthracyclines modified at benzyl ring or having C-4′-substituted-benzylated anthracyclines of the present invention issimilar to the treatment regimes of other anthracyclines and theirderivatives, although some modifications to dosage may be warranted Forexample, standard treatment with doxorubicin is described in Remington'sPharmaceutical Sciences as follows.

Doxorubicin is administered intravenously to adults at 60 to 75 mg/m² at21-day intervals or 25 to 30 mg/m² on each of 2 or 3 successive daysrepeated at 3- or 4-week intervals or 20 mg/m² once a week. The lowestdose should be used in elderly patients, when there is priorchemotherapy or neoplastic marrow invasion or when the drug is combinedwith other myelopoietic suppressant drugs. The dose should be reduced by50% if the serum bilirubin lies between 1.2 and 3 mg/dL and by 75% ifabove 3 mg/dL. The lifetime total dose should not exceed 550 mg/m² inpatients with normal heart function and 400 mg/m² in patients withabnormal heart function and 400 mg/m² on each of 3 consecutive days,repeated every 4 weeks. Prescribing limits are as with adults. It hasbeen reported that a 96-hour continuous infusion is as effective as andmuch less toxic than the same dose given by golus injections.

Of course, modifications of the treatment regimes due to the uniquenature of the substituted anthracyclines having C-3′-alkylatedanthracyclines modified at benzyl ring and having C-4′-substitutedanthracyclines of the present invention are possible and well within theability of one skilled in the art. Appropriate modifications may beascertained by following the protocols in the following examples for invivo testing and developments of human protocols.

EXAMPLE 6 In Vivo Prevention of Tumor Development Using Analogs ofAnthracyclines with Substitutions at C-3′ or C-4′ Sugars

In an initial round of in vivo trials, a mouse model of human cancerwith the histologic features and metastatic potential resembling tumorsseen in humans (Katsumata et al., 1995) is used. The animals are treatedwith analogs of anthracyclines having C-3′-substitutions as described oranthracycline analogs with C-4′-substitutions synthesized in the presentinvention to determine the suppression of tumor development.

These novel analogs are tested in vivo for anti-tumor activity againstmurine leukemia L1210, P388, and P388 resistant to doxorubicin. Inconjunction with these studies, the acute and sub-acute toxicity isstudied in mice (LD10, LD50, LD90). In a more advanced phase of testing,the anti-tumor activity of substituted anthracyclines synthesized in thepresent invention, are tested against human xenografts is assessed andcardiotoxicity studies performed is done in a rat or rabbit model.

These studies are based on the discovery that these analogs ofanthracyclines have anti-cancer activity for MDR cancer cells. Theinvention provides a useful preventive and therapeutic regimen forpatients with MDR tumors.

Two groups of mice of a suitable cancer model are treated with doses ofsubstituted anthracyclines with substitutions at the C-3′-sugar orhaving C-4′-substituted sugar moietied. Several combinations andconcentrations of the substituted anthracyclines are tested. Controlmice are treated with buffer only.

The effect of substituted anthracyclines on the development of breasttumors is compared with the control group by examination of tumor sizeand histopathologic examination (breast tissue is cut and stained withhematoxylin and eosin) of breast tissue. With the chemopreventivepotential of WP831, WP791, WP790, WP787, WP786, WP785, WP784, WP780,WP778, WP775, WP774, WP758, WP757, WP756, WP755, WP799, WP797, WP794,WP783, WP750, WP744, WP727, WP571, WP418 and WP417, it is predictedthat, unlike the control group of mice that develop tumors, the testinggroup of mice is resistant to tumor development.

EXAMPLE 7 Human Treatment with Analogs of Anthracyclines withSubstitutions at C-3′ or C-4′ Sugars

This example describes a protocol to facilitate the treatment of cancerusing substituted anthracyclines having C-3′ alkylated anthracyclinesmodified at benzyl ring and having 4′-substituted-benzylatedanthracyclines .

A cancer patient presenting, for example, an MDR cancer is treated usingthe following protocol. Patients may, but need not, have receivedprevious chemo- radio- or gene therapeutic treatments. Optimally, thepatient exhibits adequate bone marrow function (defined as peripheralabsolute granulocyte count of >2,000/mm3 and platelet count of100,000/mm3, adequate liver function (bilirubin 1.5 mg/dl) and adequaterenal function (creatinine 1.5 mg/dl)).

Exemplary Protocol for the Treatment of Multi-Drug Resistant Cancer

A composition of the present invention is typically administered orallyor parenterally in dosage unit formulations containing standard, wellknown, non-toxic physiologically acceptable carriers, adjuvants, andvehicles as desired. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intra-arterialinjection, or infusion techniques. The analogs of anthracyclinessynthesized in the present invention may be delivered to the patientbefore, after, or concurrently with the other anti-cancer agents.

A typical treatment course may comprise about six doses delivered over a7- to 21-day period. Upon election by the clinician, the regimen may becontinued six doses every three weeks or on a less frequent (monthly,bimonthly, quarterly etc.) basis. Of course, these are only exemplarytimes for treatment, and the skilled practitioner will readily recognizethat many other time-courses are possible.

A major challenge in clinical oncology is that many cancers aremulti-drug resistant. One goal of the inventors' efforts has been tofind ways to improve the efficacy of chemotherapy. In the context of thepresent invention, the substituted anthracyclines have a surprisingcytotoxicity against such cancers.

To kill MDR cancer cells using the methods and compositions described inthe present invention, one will generally contact a target cell with abisanthracycline of the present invention. These compositions areprovided in an amount effective to kill or inhibit the proliferation ofthe cell.

In certain embodiments, it is contemplated that one would contact thecell with agent(s) of the present invention about every 6 hours to aboutevery one week. In some situations, however, it may be desirable toextend the time period for treatment significantly where several days(2, 3, 4, 5, 6, 7 or more) to several weeks (1, 2, 3, 4, 5, 6, 7, ormore) lapse between respective administrations.

Regional delivery of anthracycline analogs is an efficient method fordelivering a therapeutically effective dose to counteract the clinicaldisease. Likewise, the chemotherapy may be directed to a particularaffected region. Alternatively, systemic delivery of active agents maybe appropriate.

The therapeutic composition of the present invention is administered tothe patient directly at the site of the tumor. This is in essence atopical treatment of the surface of the cancer. The volume of thecomposition should usually be sufficient to ensure that the tumor iscontacted by the substituted anthracyclines.

In one embodiment, administration simply entails injection of thetherapeutic composition into the tumor. In another embodiment, acatheter is inserted into the site of the tumor, and the cavity may becontinuously perfused for a desired period of time.

Clinical responses may be defined by acceptable measure. For example, acomplete response may be defined by the disappearance of all measurabledisease for at least a month, whereas a partial response may be definedby a 50% or greater reduction of the sum of the products ofperpendicular diameters of all evaluable tumor nodules or at least onemonth with no tumor sites showing enlargement. Similarly, a mixedresponse may be defined by a reduction of the product of perpendiculardiameters of all measurable lesions by 50% or greater, with progressionin one or more sites.

Of course, the above-described treatment regimes may be altered inaccordance with the knowledge gained from clinical trials such as thosedescribed in Example 7. Those of skill in the art are able to take theinformation disclosed in this specification and optimize treatmentregimes based on the clinical trials described in the specification.

EXAMPLE 8 Clinical Trials with Analogs of Anthracyclines withSubstitutions at C-3′ or C-4′ on the Sugar Moiety

This example is concerned with the development of human treatmentprotocols using the substituted anthracyclines. These compounds are ofuse in the clinical treatment of various MDR cancers in whichtransformed or cancerous cells play a role. Such treatment is aparticularly useful tool in anti-tumor therapy, for example, in treatingpatients with ovarian, breast and lung cancers that are resistant toconventional chemotherapeutic regimens.

The various elements of conducting a clinical trial, including patienttreatment and monitoring, is known to those of skill in the art in lightof the present disclosure. The following information is being presentedas a general guideline for use in establishing substitutedanthracyclines drugs made by the use of this invention, in clinicaltrials.

Patients with human metastatic breast and/or epithelial ovariancarcinoma, colon cancer leukemia, or sarcoma are chosen for clinicalstudy. Measurable disease is not required, however the patient must haveeasily accessible pleural effusion and/or ascites. Further the patientsmust carry tumors that express MDR phenotype. In an exemplary clinicalprotocol, patients may undergo placement of a Tenckhoff catheter, orother suitable device, in the pleural or peritoneal cavity and undergoserial sampling of pleural/peritoneal effusion. Typically, one will wishto determine the absence of known loculation of the pleural orperitoneal cavity, creatinine levels that are below 2 mg/dl, andbilirubin levels that are below 2 mg/dl. The patient should exhibit anormal coagulation profile.

In regard to the substituted anthracyclines drug administration, aTenckhoff catheter, or alternative device, may be placed in the pleuralcavity or in the peritoneal cavity, unless such a device is already inplace from prior surgery. A sample of pleural or peritoneal fluid can beobtained, so that baseline cellularity, cytology, LDH, and appropriatemarkers in the fluid (CEA, CA15-3, CA 125, p185) and in the cells (E1A,p185) may be assessed and recorded.

In the same procedure, substituted anthracyclines may be administered.The administration may be in the pleural/peritoneal cavity, directlyinto the tumor, or in a systemic manner. The starting dose may be 0.5mg/kg body weight. Three patients may be treated at each dose level inthe absence of grade>3 toxicity. Dose escalation may be done by 100%increments (0.5 mg, 1 mg, 2 mg, 4 mg) until drug related Grade IItoxicity is detected. Thereafter, dose escalation may proceed by 25%increments. The administered dose may be fractionated equally into twoinfusions, separated by 6 hours if the combined endotoxin levelsdetermined for the lot of bisanthracycline exceed 5EU/kg for any givenpatient.

The substituted anthracyclines may be administered over a short infusiontime or at a steady rate of infusion over a 7- to 21-day period. Thebisanthracycline infusion may be administered alone or in combinationwith the anti-cancer drug. The infusion given at any dose level isdependent upon the toxicity achieved after each. Hence, if Grade IItoxicity was reached after any single infusion, or at a particularperiod of time for a steady rate infusion, further doses should bewithheld or the steady rate infusion stopped unless toxicity improves.Increasing doses of substituted anthracyclines in combination with ananti-cancer drug is administered to groups of patients untilapproximately 60% of patients show unacceptable Grade III or IV toxicityin any category. Doses that are ⅔ of this value could be defined as thesafe dose.

Physical examination, tumor measurements, and laboratory tests should,of course, be performed before treatment and at intervals of about 3-4weeks later. Laboratory studies should include CBC, differential andplatelet count, urinalysis, SMA-12-100 (liver and renal function tests),coagulation profile, and any other appropriate chemistry studies todetermine the extent of disease, or determine the cause of existingsymptoms. Also appropriate biological markers in serum should bemonitored, e.g. CEA, CA 15-3, p185 for breast cancer, and CA 125, p185for ovarian cancer.

To monitor disease course and evaluate the anti-tumor responses, it iscontemplated that the patients should be examined for appropriate tumormarkers every 4 weeks, if initially abnormal, with twice weekly CBC,differential and platelet count for the 4 weeks; then, if nomyelosuppression has been observed, weekly. If any patient has prolongedmyelosuppression, a bone marrow examination is advised to rule out thepossibility of tumor invasion of the marrow as the cause ofpancytopenia. Coagulation profile shall be obtained every 4 weeks. AnSMA-12-100 shall be performed weekly. Pleural/peritoneal effusion may besampled 72 hours after the first dose, weekly thereafter for the firsttwo courses, then every 4 weeks until progression or off study.Cellularity, cytology, LDH, and appropriate markers in the fluid (CEA,CA15-3, CA 125, p185) and in the cells (p185) may be assessed. For anexample of an evaluation profile, see Table 3. When measurable diseaseis present, tumor measurements are to be recorded every 4 weeks.Appropriate radiological studies should be repeated every 8 weeks toevaluate tumor response. Spirometry and DLCO may be repeated 4 and 8weeks after initiation of therapy and at the time study participationends. A urinalysis may be performed every 4 weeks.

Clinical responses may be defined by acceptable measure. For example, acomplete response may be defined by the disappearance of all measurabledisease for at least a month. Whereas a partial response may be definedby a 50% or greater reduction of the sum of the products ofperpendicular diameters of all evaluable tumor nodules or at least onemonth with no tumor sites showing enlargement. Similarly, a mixedresponse may be defined by a reduction of the product of perpendiculardiameters of all measurable lesions by 50% or greater with progressionin one or more sites. TABLE 5 EVALUATIONS BEFORE AND DURING THERAPYTWICE EVERY 4 EVERY 8 EVALUATIONS PRE-STUDY WEEKLY WEEKLY WEEKS WEEKSHistory X X Physical X X Tumor Measurements X X CBC X X¹ X DifferentialX X¹ X Platelet Count X X¹ X SMA12-100 (SGPT, Alkaline X X Phosphatase,Bilirubin, Alb/Total Protein) Coagulation Profile X X Serum Tumormarkers X  X³ (CEA, CA15-3, CA-125, Her-2/neu) Urinalysis X X X-rays:chest X  X⁴ others X X Pleural/Peritoneal X  X⁵ X Fluids: (cellularity,cytology, LDH, tumor markers, E1A, HER-2/neu) Spirometry and DLCO X  X⁶ X⁶¹For the first 4 weeks, then weekly, if no myelosuppression is observed.²As indicated by the patient's condition.³Repeated every 4 weeks if initially abnormal.⁴For patients with pleural effusion, chest X-rays may be performed at 72hours after first dose, then prior to each treatment administration.⁵Fluids may be assessed 72 hours after the first dose, weekly for thefirst two courses and then every 4 weeks thereafter.⁶Four and eight weeks after initiation of therapy.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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U.S. Pat. No. 4,263,428 Apr. 21, 1981.

U.S. Pat. No. 4,345,070, Aug. 17, 1982.

U.S. Pat. No. 4,438,105, Mar. 20, 1984.

1-47. (canceled)
 48. A method of treating cancer comprisingadministering to a patient a DNA damaging agent and a substitutedanthracycline having the formula:

wherein R is a H or OCH₃.
 49. The method of claim 48, wherein R is H.50. The method of claim 48, wherein R is OCH₃.
 51. The method of claim48, wherein the DNA damaging agent is a chemotherapeutic agent.
 52. Themethod of claim 51, wherein the chemotherapeutic agent is selected fromthe group consisting of 5-fluorouracil (5FU), etoposide (VP-16),camptothecin, actinomycin-D, mitomycin, cisplatin (CDDP), hydrogenperoxide, carboplatin, procarbazine, mechlorethamine, cyclophosphamide,ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin,bleomycin, plicomycin, tamoxifen, taxol, transplatinum, vincristine,vinblastine, and methotrexate.
 53. The method of claim 48, wherein theDNA damaging agent is a radiotherapeutic agent.
 54. The method of claim53, wherein the radiotherapeutic agent is radiation.
 55. The method ofclaim 54, wherein the radiation is selected from the group consisting ofγ-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions,and radioisotopes.
 56. The method of claim 48, wherein the cancer isbreast cancer, lung cancer, ovarian cancer, Hodgkin's disease,non-Hodgkin's lymphoma, acute leukemia, or carcinoma of the testes. 57.The method of claim 48, wherein the substituted anthracycline isformulated for parenteral administration.
 58. The method of claim 48,wherein the substituted anthracycline is formulated for oraladministration.
 59. The method of claim 48, wherein the DNA damagingagent is formulated for parenteral administration.
 60. The method ofclaim 48, wherein the DNA damaging agent is formulated for oraladministration.
 61. The method of claim 48, wherein the substitutedanthracycline is administered before the DNA damaging agent isadministered to the patient.
 62. The method of claim 48, wherein thesubstituted anthracycline is administered at the same time the DNAdamaging agent is administered to the patient.
 63. The method of claim48, wherein the substituted anthracycline is administered after the DNAdamaging agent is administered to the patient.
 64. The method of claim48, wherein the DNA damaging agent is administered before thesubstituted anthracycline is administered to the patient.
 65. The methodof claim 48, wherein the DNA damaging agent is administered at the sametime the substituted anthracycline is administered to the patient. 66.The method of claim 48, wherein the DNA damaging agent is administeredafter the substituted anthracycline is administered to the patient. 67.The method of claim 48, wherein the substituted anthracycline and theDNA damaging agent are formulated into a single composition.
 68. Apharmaceutically acceptable composition comprising a DNA damaging agentand a substituted anthracycline having the formula:

wherein R is a H or OCH₃.
 69. The pharmaceutically acceptablecomposition of claim 68, wherein R is H.
 70. The pharmaceuticallyacceptable composition of claim 68, wherein R is OCH₃.
 71. Thepharmaceutically acceptable composition of claim 68, wherein the DNAdamaging agent is a chemotherapeutic agent.
 72. The pharmaceuticallyacceptable composition of claim 71, wherein the chemotherapeutic agentis selected from the group consisting of 5-fluorouracil (5FU), etoposide(VP-16), camptothecin, actinomycin-D, mitomycin, cisplatin (CDDP),hydrogen peroxide, carboplatin, procarbazine, mechlorethamine,cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan,nitrosurea, doxorubicin, bleomycin, plicomycin, tamoxifen, taxol,transplatinum, vincristine, vinblastine, and methotrexate.
 73. Thepharmaceutically acceptable composition of claim 68, wherein thecomposition is formulated for parenteral administration.
 74. Thepharmaceutically acceptable composition of claim 68, wherein thecomposition is formulated for oral administration.
 75. A method oftreating cancer comprising administering to a patient a DNA damagingagent and a substituted anthracycline having the formula:

wherein, R¹ is an alkyl chain, a (—COCH₂R¹³) group, or a (C(OH)—CH₂R¹³)group; wherein, R¹³ is a hydrogen (—H) group, a hydroxyl group (—OH), amethoxy group (—OCH ₃), an alkoxy group comprising 1-20 carbon atoms, analkyl group comprising 1-20 carbon atoms, an aryl group comprising 1-20carbon atoms, a fatty acyl group comprising the general structure—O—CO(CH₂)_(n)CH₃, wherein n=an integer from 1 to about 20, a fatty acylgroup comprising the general structure—O—CO(CH₂)_(l)(CH═CH)_(m)(CH₂)_(n)CH₃, a —OCO—(CH₂)_(n)—CH₂NH₂ group, ora —OCO—(CH₂)_(n)—CO₂H group, wherein 1 is an integer between 1 to 3, mis an integer between 1 and 6, and n is an integer between 1 and 9;wherein R² and R³ are, independently of the other, a hydrogen (—H), ahydroxyl group (—OH), or a methoxy group (—OCH₃); wherein R⁴ is ahydrogen (—H) group, a methoxy group (—OCH₃), a hydroxyl group (—OH), ora halide; wherein Y¹ and Y² are, independently of the other, a doublebonded oxygen, sulphur, or nitrogen atom; wherein Z is a —H, —OH, a—CO₂H, or a —CO₂R group; wherein R⁵ and R⁶, are, independently, —H, —OH,a halide, —OR¹⁹, —SH, —SR¹⁹, —NH₂, —NHR¹⁹, —N(R¹⁹)₂ or —CH₃, and R⁵ canadditionally be an alkylating moiety, wherein R¹⁹ is an alkyl chain, analkylating moiety, a cycloalkyl chain, a cyclic ring, or a hydrogen;wherein R⁹ is an —H, —CH₃, alkyl, aryl, CH₂OH, or CH₂F group; whereinR¹⁰, R¹¹, and R¹² are, independently, —H, —OH, a halide, —OR, —SH, —SR,—NH₂, —NHR, —N(R)₂ or —CH₃; wherein one of R⁷ and R⁸ is an —H andwherein one of R⁷ and R⁸ is a X-alkyl aromatic-ring (—XAAR) substituent,wherein, A is an alkyl group and wherein, AR is an unsubstituted phenylring, a substituted phenyl ring, a substituted five-member ring or aheteroatomic five-member ring, of the general form:

wherein, R¹⁴-R¹⁸ are independently a (—H) group, a hydroxyl group (—OH),a methoxy group (—OCH₃), a nitro group (—NO₂), an amine group (—NH₂), ahalide, an alkoxy group having 1-20 carbon atoms, an alkyl group having1-20 carbon atoms, an aryl group having 1-20 carbon atoms, analkyl-amino group, an alkyl-thio group, a cyano group (CN, SCN), an—CO₂H group, or a —CO₂R group; and X is a —O, —N, —S, —SO, or a —SO₂group; and A is (CH₂)_(n), where n=0-10; wherein if R⁷ is a XAARsubstituent R⁸is not and if R⁸ is a XAAR substituent R⁷ is not.
 76. Themethod of claim 75, wherein the aromatic ring of the —XAAR substituentis disubstituted, trisubstituted, tetrasubstituted, or pentasubstituted.77. The method of claim 75, wherein the DNA damaging agent is achemotherapeutic agent.
 78. The method of claim 77, wherein thechemotherapeutic agent is selected from the group consisting of5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D,mitomycin, cisplatin (CDDP), hydrogen peroxide, carboplatin,procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan,chlorambucil, bisulfan, nitrosurea, dactinomycin, bleomycin, plicomycin,tamoxifen, taxol, transplatinum, vincristine, vinblastine, andmethotrexate.
 79. The method of claim 75, wherein the DNA damaging agentis a radiotherapeutic agent.
 80. The method of claim 75, wherein theradiotherapeutic agent is radiation.
 81. The method of claim 80, whereinthe radiation is selected from the group consisting of γ-irradiation,X-rays, UV-irradiation, microwaves, electronic emissions, andradioisotopes.